Recombinant antibodies against infectious bursal disease virus (ibdv)

ABSTRACT

The purpose of this project was to isolate recombinant antibodies for diagnosis of very virulent infectious bursal disease virus (vvIBDV) in fixed tissues. Phage-displayed recombinant antibodies, comprised of the single chain variable fragment (scFv), were investigated. A previously made recombinant antibody library generated against vvIBDV was selected and screened for recombinant antibodies that reacted against vvIBDV in ELISA. A new library was constructed fom chickens immunized with fixed vvIBDV and also screened for recombinant antibodies against vvIBDV. Also, a previously identified recombinant antibody, known to react well with vvIBDV, was used to replace either the Vh or VI gene with corresponding fragments from a new library. The Vh and VI antibody genes were initially amplified effectively by PCR. No new recombinant antibody clones were isolated from the libraries generated against vvIBDV. However, exchanging the Vh or VI genes from a known recombinant antibody with genes from a new library showed that the heavy chain was essential when binding to vvBDV. Light chains could be exchanged without loss of activity, but when heavy chains were exchanged, all activity was lost. The light chains were found to create new binding properties when combined with the essential heavy chain. The recombinant antibody clones were sequenced, analyszd and characterize.

FIELD OF THE INVENTION

The present invention relates to the field of recombinant antibodies,and more particularly to the generation and screening of recombinantantibodies generated against infectious bursal disease virus (IBDV). Thepresent invention also provides neutralising antibodies against IBDV andvarious means by which a wide range of IBDV recombinant antibody-basedtherapeutics, prophylactics and diagnostic reagents may be developed.

BACKGROUND

Infectious bursal disease virus (IBDV) belongs to the Birnaviridaefamily. It causes a highly contagious immunosuppressive disease inchickens by depleting B cell populations within the Bursa of Fabricius.

The IBDV virion consists of a double stranded RNA genome in anon-enveloped icosahedral capsid. The capsid contains two majorstructural proteins, VP3 and VP2. Virus neutralizing antibodies havebeen shown to recognise a conformational epitope located in thehypervariable region of VP2, between amino acid residues 206 and 350.Thus, the VP2 protein is an important target for immunodetection andimmunoprophylaxis studies.

A large number of IBDV strains have been identified throughout the worldand grouped according to their relative pathogenicity and antigenicity.Three major groups have been identified: (i) classical strains, (ii)very virulent types (vvIBDV) capable of causing up to 70% mortality and(iii) antigenic variants. In Australia both classical and variant fieldstrains of IBDV have been identified which are genetically distinct fromoverseas strains. Australian classical strains are similar to vaccinestrains such as 002/73 and V877 both antigenically and by nucleotidesequencing. Australian variants, however, are quite distinct both at theantigenic and genetic level forming a distinct group of viruses that areunrelated to variants isolated in other countries such as the USA.

The ability to differentiate and treat infection caused by IBDV strainsis a major concern to the poultry industry worldwide, as effectivecontrol of this devastating disease relies on the administration ofdifferent vaccine strains. In many countries both accidental incursionof IBDV strains from neighbouring countries and the absence of a testfor establishing whether outbreaks in the field are due to vaccinebreaks or circulating field strains are also major problems.

Differentiation of IBDV strains is currently largely based upon usingeither monoclonal antibodies directed against the major neutralisingprotein of the virus (VP2) or by nucleotide sequencing of the VP2 gene.Monoclonal antibodies generated against the VP2 and VP3 gene producthave perhaps played the most significant role in detecting anddifferentiating IBDV strains and several VP2 and VP3 specific monoclonalantibodies have been developed.

Testing with monoclonal antibodies is generally quicker andsignificantly cheaper than other prior art technologies, however,monoclonal antibodies are limited in terms of their differentiation.Furthermore, a major drawback of using monoclonal antibodies asimmunodiagnostics or immunotherapeutic reagents lies in the cost andtime required for production, screening and maintenance of hybridomacell lines. In addition, an anti-antibody response is often induced whenmonoclonal antibodies are administered in a heterologous species, thuscompromising their effectiveness.

Thus, there is currently a need for improved diagnostic and ortherapeutic agents effective against IBDV. In particular there is a needfor diagnostics that are capable of differentiating IBDV strains, thatmight be used to track the incursion of exotic strains of IBDV into aparticular country and or for differentiating IBDV strains very similarto vaccine IBDV strains from circulating field strains. In addition,there is a need to differentiate between an immune response induced byIBDV vaccines from that induced by field strains. There is also strongdemand for new therapeutics capable of combating this devastatingdisease.

SUMMARY OF THE INVENTION

The present invention provides at least a recombinant antibody fragmentagainst IBDV. More particularly, the recombinant antibody fragment willhave specificity for an IBDV antigenic determinant and comprise avariable region having a heavy chain (V_(H)) region and or a light chain(V_(L)) region.

The subject invention also provides polynucleotides encoding specificrecombinant antibody fragments as described herein. In preferred form ofthe invention it provides nucleic acid molecules encoding V_(H) or V_(L)regions or single chain antibody fragments comprising V_(H) and V_(L)regions linked together via a linker.

The present invention also provides a process for identifyingrecombinant antibody fragments, which process comprises the steps:

-   (i) Amplifying nucleotide sequences comprising V_(H) and V_(L)    chains from lymphocytes from a host which has been caused to produce    antibodies against at least IBDV or an IBDV polypeptide or a    fragment thereof;-   (ii) Generating a library comprising amplified nucleotide sequences    from step (i), which library is capable of being screened to    identify V_(H) and or V_(L) regions reactive with at least IBDV or    an IBDV polypeptide or a fragment thereof; and-   (iii) Screening said library and selecting at least a recombinant    antibody fragment that has an affinity for IBDV or an IBDV    polypeptide or a fragment thereof.

Such a method is particularly useful for identifying recombinant IBDVantibodies that may serve as immunodiagnostic and or immunotherapeuticagents that may for example serve as candidate antagonists of IBDVbiological activity.

The isolation of recombinant antibodies from the host infected with IBDVhas the advantage that a degree of certainty is associated with theimmunological specificity of the generated antibodies. When antibodiesare produced and amplified in one animal species that is not a naturaltarget (eg a mouse system) different from the animal species that is anatural target of infection (eg a chicken) with a virus, antibodypresentation is dependant on the first host species immune system andthe way in which antigen is presented in that system. Potentially thereare many antigenic determinants that may not be recognised by the firstanimal species that are recognised by the immune system of the naturaltarget species. The method of the present invention seeks to capitaliseon this aspect of antigen presentation in that antibodies produced bythe method of the invention are derived from the infected-natural hostanimal, rather than an artificially infected species, which is not anatural target of a virus. Amplification of the antibodies is thenachieved by recombinant molecular biology techniques therein assuringthat immunological specificity within the selected antibody populationis not diluted or indeed lost as perhaps may happen when foreign(different) animal species are used to produce said antibodies.

Recombinant IBDV antibodies capable of distinguishing IBDV strains canbe selected by comparing the observed activity when such antibodies arebrought in contact with the stains in the presence of a control system.Using differences in the observed activity it is possible to identifyrecombinant IBDV antibodies which are either desired for the ability toidentify particular IBDV strains or in the alternate case those whichare not able to identify these strains.

The invention also provides a method for detecting the presence of IBDVin a sample comprising contacting said sample with a recombinant IBDVantibody which specifically binds to an IBDV antigen, comprising avariable region having a heavy chain region and a light chain region,and determining binding of said antibody to IBDV antigen in said sampleas a determination of IBDV infection in said sample.

Further the invention provides therapeutic, pharmaceutical or diagnosticcompositions comprising a recombinant antibody fragment according to theinvention, in combination with a pharmaceutically acceptable excipient,diluent or carrier.

BRIEF DESCRIPTION OF DRAWINGS

The Figures are described as follows:

FIG. 1: Sequence and restriction sites of the multiple cloning regionsof pCANTAB 5E and derived plasmids pCANTAB-Ascl, pCANTAB-Smal andpCANTAB-link

FIG. 2: Plasmid map pCANTAB-link.

FIG. 3: (FIGS. 3A to I) Nucleotide sequence of the variable regions ofthe heavy chain (SEQ ID NOS: 1-35) and (FIG. 3J to R) (light chain (SEQID NOS 36-77) genes of scFV clones.

FIG. 4: Deduced amino acid sequences of the variable regions of theheavy (FIG. 4A to E) and light chain (FIG. 4F to J) of scFv clones.

FIG. 5: Alignment of the deduced amino acid sequences of CRAbs. Onlythose amino acids that differ from the consensus sequence are shown,while identical amino acids are shown by dots and the absence ofcorresponding residues is shown by dashes. Complementarity regions areshown underlined while the linker sequence (Gly₄Ser)₃ is shown in bold.CRAb34 and CRAb0 were obtained by cloning of scFv's into pCANTAB 5Ewhile the remaining CRAbs were obtained by sequential cloning of VH andVL genes into pCANTAB-link vector.

FIG. 6: Reactivity of CRAb34 with a panel of IBDV strains in ELISA.

FIG. 7: Titration of CRAb34 soluble antibody against Australian IBDVstrains. Various doses of soluble CRAb 34 were titrated against fixedconcentrations of viral antigen coated on the microtitre plate.

FIG. 8: CRAbs compete with immune ant-IBDV sera for binding to 002/73antigen.

FIG. 9: Competition by SAbs specific for Australian (11, 34, 33, 52, 72)or overseas (28, 29, 88) IBDV strains with anti-002/73 sera for bindingto 002/73 Australian IBDV antigen

FIG. 10:. Competition by SAbs specific for Australian (11, 34, 33, 52,72) or overseas (28, 29, 88) IBDV strains with anti-very virulent CS88sera for binding to very virulent CS88 antigen

FIG. 11: Partial neutralization of BursvacL vaccine with various CRAbsin 2-week-old specific pathogen-free chickens

FIG. 12: Neutralization of various doses of BursvacL vaccine by CRAb8 inday-old or 2-week-old* chickens

FIG. 13: Time course of BursavacL release from BursavacL.CRAb8 complexfollowing inoculation into 18-day-old specific pathogen-free embryos Q

FIG. 14: Immunoprecipitation with Sab34 and Sab0 showing that CRAb34recognises the VP2 protein of 002/73 strain of IBDV

DETAILED DESCRIPTION OF THE INVENTION

General

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively, andany and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention as describedherein.

Sequence identity numbers (SEQ ID NO:) containing nucleotide and aminoacid sequence information included in this specification are collectedat the end of the description and have been prepared using the programmePatentin Version 3.1. Each nucleotide or amino acid sequence isidentified in the sequence listing by the numeric indicator <210>followed by the sequence identifier (e.g. <210>1, <210>2, etc.). Thelength, type of sequence and source organism for each nucleotide oramino acid sequence are indicated by information provided in the numericindicator fields <211>, <212> and <213>, respectively. Nucleotide andamino acid sequences referred to in the specification are defined by theinformation provided in numeric indicator field <400> followed by thesequence identifier (e.g. <400>1, <400>2, etc.).

The entire disclosures of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference. Noadmission is made that any of the references constitute prior art or arepart of the common general knowledge of those working in the field towhich this invention relates.

As used herein the term “derived” and “derived from” shall be taken toindicate that a specific integer may be obtained from a particularsource albeit not necessarily directly from that source.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other scientific and technical terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the invention belongs.

Description

The present invention provides novel, recombinant antibody fragmentsspecific for IBDV. The invention also provides methods for theproduction of these antibody fragments as well as methods for their usein the diagnosis and treatment of IBDV disease.

The term “recombinant antibody fragment” is used herein to denote anyantibody fragment produced using recombinant DNA or in vitro proteinsynthesis techniques, and excludes monoclonal antibodies produced bytraditional monoclonal antibody techniques. Preferably, such fragmentsare initially derived from nucleotide sequences encoding heavy and lightchain variable regions produced in B-lymphocytes from a host that hasbeen caused to produce antibodies against at least IBDV or an IBDVpolypeptide or a fragment thereof.

The term “antibody” as used herein, unless indicated otherwise, is usedbroadly to refer to, for example, Fv fragments, single-chain Fvfragments (scFv), Fab′ fragments, and F(ab′)2 fragments, diabodies,individual V_(L) chains, individual V_(H) chains, chimeric fusionsbetween V_(H) and or V_(L) chains and other molecules, and the like.

Recombinant antibody fragments including substitutions, deletions andmodifications within the scope of the present invention include thoseantibodies that have binding affinity for at least an IBDV strain. Inmost instances such antibodies will recognise more than, one IBDVstrain, however, where binding is specific to a single IBDV strain thatrecombinant antibody fragment may provide a means to differentiallyidentify that strain. Preferably, the recombinant antibody fragments arecapable of binding one or more of the group selected from: AustralianIBDV strain 002/73; vaccine strain V877; classical field strains 06/95,K3, M4, R1, T4, N1/99, N2/99, A-1 or Y-5; variant field strains 01/94,02/95, 03/95, 04/95 or 08/95; classical overseas strains 52/70 and 1/68;variant strain E and vvIBDV strain CS88.

Recombinant antibody fragments according to the invention may be derivedfrom any host that has been caused to produce antibodies against IBDV oran IBDV polypeptide or fragment thereof. Preferably, that host is ofavian origin. Even more preferably the host is a fowl such as forexample, chickens, turkeys, guinea fowls, ducks, and geese. In thepresent disclosure the invention is described in terms of recombinantantibody fragments derived from domestic fowl and in particularchickens. It should be appreciated however that the scope of thedisclosure is not so limited and hence the invention may apply to anyavian species that succumbs to IBDV.

The isolation of recombinant antibody fragments from domestic fowl hasthe advantage that the antibody fragments are obtained from a normalhost for IBDV. In addition, deriving recombinant antibody fragments fromdomestic fowls offer technical advantages over antibodies from othermammalian hosts like mice as the variable region genes in domestic fowlare flanked by constant regions and can easily be amplified usingtechniques such as polymerase chain reaction (PCR), using a singleprimer set.

IBDV Related Recombinant Antibody Polypeptide Fragments

It should be appreciated that the IBDV related recombinant antibodyfragment(s) described herein can take a variety of forms. Preferably therecombinant antibody fragment(s) are Fv fragments, scFv or Fab′fragments that have an affinity for IBDV.

According to the invention the V_(H) domain and the V_(L) domain withinthe recombinant antibody fragment may be linked in a single chain toproduce a single chain Fv fragment or bound by one or more covalentbonds such as disulphide bonds. Where the recombinant antibody fragmentis prepared as an scFv fragment, the V_(H) domain and the V_(L) domainare preferably linked by a short peptide spacer (usually 15-20 aminoacids long) that is introduced at the genetic level during theconstruction of the scFv. Linkage of V_(H) and V_(L) regions may beachieved by any method known in the art. For example, a synthetic linkersuch as a flexible glycine-serine linker may be used. An example of alinker that is illustrated in the Examples herein is (Gly₄Ser)₃.

In a highly preferred form of the invention the recombinant antibodyfragments comprise: a V_(H) region selected from the group consisting ofSEQ ID NOS:78 to 112, or a homologue of any one of these sequences andor a V_(L) region selected from the group consisting of SEQ ID NOS:113to 154, or a homologue of any one of these sequences.

A homologous sequence is taken to include an amino acid sequence whichis at least 60, 70, 80 or 90% identical, preferably at least 95 or 98%identical at the amino acid level over at least 20, 50 or 100 aminoacids against which that sequence is compared and will possess IBDVrelated immunological properties. Homology should typically beconsidered with respect to those regions of the sequence known to beessential for the function of the protein rather than nonessentialneighbouring sequences. Thus, for example, homology comparisons arepreferably made over V_(H) and V_(L) chain regions and more particularlyover those regions of the V_(H) and V_(L) chains that are essential forantigen binding.

Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present invention it is preferred to express homologyin terms of sequence identity. Homology comparisons can be conducted byeye, or more usually, with the aid of readily available sequencecomparison programs. These commercially available computer programs cancalculate % homology between two or more sequences.

It will be appreciated that any V_(H) may be linked to any V_(L) region.By pairing different V_(H) and V_(L) regions to produce recombinantantibody fragments it is possible to change the immunological profile ofthese fragments. Thus, when referring to the sequences exemplified inthis application any of SEQ ID NOS:1 to 35 may be linked to any of SEQID NOS:36 to 77. Preferably, however, the sequences are linked via asynthetic linker like (Gly₄Ser)₃ in the following order:

-   -   SEQ ID NO:1 linked to SEQ ID NO:60; [SEQ ID NO:155 (CRAb19)]    -   SEQ ID NO:1 linked to SEQ ID NO:61; [SEQ ID NO:156 (CRAb 83)]    -   SEQ ID NO:1 linked to SEQ ID NO:63; [SEQ ID NO:157 (CRAb 03)]    -   SEQ ID NO:1 linked to SEQ ID NO:64; [SEQ ID NO:158 (CRAb 24)]    -   SEQ ID NO:1 linked to SEQ ID NO:65; [SEQ ID NO:159 (CRAb 15)]    -   SEQ ID NO:1 linked to SEQ ID NO:66; [SEQ ID NO:160 (CRAb 05)]    -   SEQ ID NO:1 linked to SEQ ID NO: 67; [SEQ ID NO:161 (CRAb 33)]    -   SEQ ID NO:1 linked to SEQ ID NO: 68; [SEQ ID NO:162 (CRAb 96)]    -   SEQ ID NO:2 linked to SEQ ID NO: 62; [SEQ ID NO:163 (CRAb 34)]    -   SEQ ID NO:3 linked to SEQ ID NO: 36; [SEQ ID NO:164 (CRAb 18)]    -   SEQ ID NO:4 linked to SEQ ID NO. 37; [SEQ ID NO:165 (CRAb 37)]    -   SEQ ID NO:5 linked to SEQ ID NO: 38; [SEQ ID NO:166 (CRAb 32)]    -   SEQ ID NO:6 linked to SEQ ID NO: 39; [SEQ ID NO:167 (CRAb 66)]    -   SEQ ID NO:7 linked to SEQ ID NO: 40; [SEQ ID NO:168 (CRAb 04)]    -   SEQ ID NO:8 linked to SEQ ID NO; 41; [SEQ ID NO:169 (CRAb 62)]    -   SEQ ID NO:9 linked to SEQ ID NO: 42; [SEQ ID NO:170 (CRAb 45)]    -   SEQ ID NO:10 linked to SEQ ID NO: 43; [SEQ ID NO:171 (CRAb 149)]    -   SEQ ID NO:11 linked to SEQ ID NO: 44; [SEQ ID NO:172 (CRAb 154)]    -   SEQ ID NO:12 linked to SEQ ID NO:45; [SEQ ID NO:173 (CRAb 151)]    -   SEQ ID NO:13 linked to SEQ ID NO: 46; [SEQ ID NO:174 (CRAb 176)]    -   SEQ ID NO:14 linked to SEQ ID NO: 47; [SEQ ID NO:175 (CRAb 28)]    -   SEQ ID NO:15 linked to SEQ ID NO: 48; [SEQ ID NO:176 (CRAb 88)]    -   SEQ ID NO:16 linked to SEQ ID NO: 49; [SEQ ID NO:177 (CRAb 29)]    -   SEQ ID NO:17 linked to SEQ ID NO: 50; [SEQ ID NO:178 (CRAb 50)]    -   SEQ ID NO:18 linked to SEQ ID NO: 51; [SEQ ID NO:179 (CRAb 38)]    -   SEQ ID NO:19 linked to SEQ ID NO: 52; [SEQ ID NO:180 (CRAb 30)]    -   SEQ ID NO:20 linked to SEQ ID NO: 53; [SEQ ID NO:181 (CRAb 39)]    -   SEQ ID NO:21 linked to SEQ ID NO: 54; [SEQ ID NO:182 (CRAb 27)]    -   SEQ ID NO:22 linked to SEQ ID NO: 55; [SEQ ID NO:183 (CRAb 35)]    -   SEQ ID NO:23 linked to SEQ ID NO: 56; [SEQ ID NO:184 (CRAb 174)]    -   SEQ ID NO:24 linked to SEQ ID NO: 57; [SEQ ID NO:185 (CRAb 26)]    -   SEQ ID NO:25 linked to SEQ ID NO: 58; [SEQ ID NO:186 (CRAb 7)]    -   SEQ ID NO:26 linked to SEQ ID NO: 59; [SEQ ID NO:187 (CRAb 12)]    -   SEQ ID NO:27 linked to SEQ ID NO: 69; [SEQ ID NO:188 (CRAb 23)]    -   SEQ ID NO:28 linked to SEQ ID NO: 70; [SEQ ID NO:189 (CRAb 22)]    -   SEQ ID NO:29 linked to SEQ ID NO: 71; [SEQ ID NO:190 (CRAb 52)]    -   SEQ ID NO:30 linked to SEQ ID NO: 72; [SEQ ID NO:191 (CRAb 08)]    -   SEQ ID NO:31 linked to SEQ ID NO: 73; [SEQ ID NO:192 (CRAb 11)]    -   SEQ ID NO:32 linked to SEQ ID NO: 74; [SEQ ID NO:193 (CRAb 21)]    -   SEQ ID NO:33 linked to SEQ ID NO: 75; [SEQ ID NO:194 (CRAb 9)]    -   SEQ ID NO:34 linked to SEQ ID NO: 76; [SEQ ID NO:195 (CRAb 20)]    -   SEQ ID NO:35 linked to SEQ ID NO: 77; [SEQ ID NO:196 (CRAb 0)]

In a highly preferred form of the invention the recombinant antibodyfragment is selected from the group comprising: SEQ ID NO:155 to SEQ IDNO:196, or is a homologue of any one of these sequences.

It will be appreciated that the amino acid sequences for V_(H) and V_(L)regions described herein may also be modified in any manner or form thatdoes not extinguish and more preferably substantially alter the affinityof V_(H) and V_(L) regions to an IBDV antigen. Such modifications may benaturally and non-naturally occurring. By way of example, themodifications may include, deletions, additions, substitutions,glycosylations, acetylations, phosphorylations, and the like. Examplesof amino acid sequence substitution modifications that may be made torecombinant antibody fragments include: (a) one or more aspartic acidresidues is substituted with glutamic acid; (b) one or more isoleucineresidues is substituted with leucine; (c) one or more glycine or valineresidues is substituted with alanine; (d) one or more arginine residuesis substituted with histidine; or (e) one or more tyrosine orphenylalanine residues is substituted with tryptophan.

Recombinant antibody fragments in lacking glycosylation and the regionscomprising the binding sites for complement and Fc-receptors, also lackthe natural effector function associated with these regions. Severalstrategies have been developed to reintroduce these natural effectorfunctions into recombinant antibodies, e.g. the generation ofbi-specific antibody fragments for recruitment of effector molecules andcells. For example, IBDV related recombinant antibody fragments may befused with polypeptide sequences expressing different effectorfunctions, like toxins, enzymes, cytokines, reporter genes (fordiagnostic and imaging applications) and the like. Such fragments arenot only useful for IBDV diagnosis, but find much greater applicationsin IBDV immunotherapy and gene therapy. For IBDV therapy, an advantageof recombinant antibody fragments is their small size (the size of ascFv is only about 25 kD), facilitating tissue penetration,bio-distribution and blood clearance. However, it has been shown thatsomewhat larger fragments (50-80 kD) show in some cases even betterpharmacokinetics and that di- or multivalent fragments increase thefunctional affinity and thereby tissue targeting. Recombinant antibodyfragments can furthermore be easily used as building blocks for geneticengineering of new effector mechanisms, affinity maturation, andhumanisation.

Therefore, according to another embodiment in the invention therecombinant antibody fragments described may be conjugated with, orattached to other antibodies (or parts thereof) such as monoclonalantibodies. These other antibodies may be reactive with other markers(epitopes) characteristic for the disease against which the antibodiesof the invention are directed or may have different specificitieschosen, for example, to recruit fragments or cells of the animals immunesystem to the diseased cells. The antibodies of the invention (or partsthereof) may be linked to such antibodies by conventional chemical or bymolecular biological methods.

According to a further aspect of the invention there is provided amultivalent monospecific recombinant antibody fragment comprising two,three, four or more single chain antibody fragments or fragments thereofbound to each other by a connecting structure which protein is not anatural immunoglobulin, each of said recombinant antibody, fragments orfragments having a specificity for an IBDV epitope said protein beingoptionally conjugated with an effector or reporter fragment.

IBDV Related Recombinant Antibody Polynucleotide Sequences

Determination of the amino acid sequence for a recombinant antibodyfragment will reveal information about the likely nucleotide sequenceencoding that fragment. Using that information the nucleotide sequencefor the recombinant fragment may be obtained. Once the nucleotidesequence for a recombinant antibody fragment has been identified its DNAsequences may be synthesised completely or in part using standardoligonucleotide synthesis techniques. Site-directed mutagenesis andpolymerase chain reaction (PCR) techniques may be used as appropriate tomodify and or amplify such sequences.

Thus, the subject invention provides also polynucleotides encodingspecific recombinant antibody fragments as described herein. The subjectpolypeptides may be encoded by a wide variety of sequences because ofthe degeneracy of the genetic code. A person of ordinary skill in theart may readily change a given polynucleotide sequence encoding an IBDVspecific antibody into a different polynucleotide encoding the same IBDVspecific antibody embodiment. The polynucleotide sequence encoding theantibody may be varied to take into account factors affecting expressionsuch as codon frequency, RNA secondary structure, and the like.

According to the invention there is provided a nucleic acid fragmentencoding an isolated recombinant antibody fragment or an allelic variantor analogue or fragments thereof, which is capable of specificallybinding IBDV. Specifically provided are DNA molecules encoding V_(H) orV_(L) regions or single chain antibody fragments comprising V_(H) andV_(L) regions linked together via a linker. Most preferably, thenucleotide sequences are selected from the group consisting of: (a) DNAmolecules set out in SEQ ID NOS:1 to 77 or fragments thereof; (b) DNAmolecules that hybridise to the DNA molecules defined in (a) orhybridisable fragments thereof; and (c) DNA molecules that encodeexpression for the amino acid sequence encoded by any of the foregoingDNA molecules.

Preferred DNA molecules according to the invention include DNA moleculescomprising the sequence set out in SEQ ID NOS:1 to 77 or fragmentsthereof as well as nucleic acid sequences that are substantiallyhomologous to these sequences.

A polynucleotide is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA forand/or the polypeptide or a fragment thereof. The anti-sense strand isthe complement of such a nucleic acid, and the encoding sequence can bededuced there from.

An “isolated” or “substantially pure” nucleic acid (e.g., an RNA, DNA ora mixed polymer) is one which is substantially separated from othercellular components which naturally accompany a native animal sequenceor protein, e.g., ribosomes, polymerases, many other animal genomesequences and proteins. The term embraces a nucleic acid sequence orprotein that has been removed from its naturally occurring environment,and includes recombinant or cloned DNA isolates and chemicallysynthesized analogues or analogues biologically synthesized byheterologous systems.

A nucleic acid or fragment thereof is “substantially homologous” (“orsubstantially similar”) to another if, when optimally aligned (withappropriate nucleotide insertions or deletions) with the other nucleicacid (or its complementary strand), there is nucleotide sequenceidentity in at least about 60% of the nucleotide bases, usually at leastabout 70%, more usually at least about 80%, preferably at least about90%, and more preferably at least about 95-98% of the nucleotide bases.

Alternatively, substantial homology or (identity) exists when a nucleicacid or fragment thereof will hybridise to another nucleic acid (or acomplementary strand thereof) under selective hybridisation conditions,to a strand, or to its complement. Selectivity of hybridisation existswhen hybridisation that is substantially more selective than total lackof specificity occurs. Typically, selective hybridisation will occurwhen there is at least about 55% identity over a stretch of at leastabout 14 nucleotides, preferably at least about 65%, more preferably atleast about 75%, and most preferably at least about 90%. The length ofhomology comparison, as described, may be over longer stretches, and incertain embodiments will often be over a stretch of at least about ninenucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides.

Thus, polynucleotides of the invention preferably have at least 75%,more preferably at least 85%, more preferably at least 90% homology tothe sequences shown in the sequence listings herein. More preferablythere is at least 95%, more preferably at least 98%, homology.Nucleotide homology comparisons may be conducted as described below forpolypeptides. A preferred sequence comparison program is the GCGWisconsin Bestfit program. The default scoring matrix has a match valueof 10 for each identical nucleotide and −9 for each mismatch. Thedefault gap creation penalty is −50 and the default gap extensionpenalty is −3 for each nucleotide.

In the context of the present invention, a homologous sequence is takento include a nucleotide sequence which is at least 60, 70, 80 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel over at least 20, 50, 100, 200, or 300 nucleotides with thenucleotides sequences set out in SEQ ID NOS: 1 to 77.

Generally, the shorter the length of the polynucleotide, the greater thehomology required to obtain selective hybridisation. Consequently, wherea polynucleotide of the invention consists of less than about 30nucleotides, it is preferred that the % identity is greater than 75%,preferably greater than 90% or 95% compared with the recombinantantibody fragment nucleotide sequences set out in the sequence listingsherein. Conversely, where a polynucleotide of the invention consists of,for example, greater than 50 or 100 nucleotides, the % identity comparedwith the nucleotide sequences set out in the sequence listings hereinmay be lower, for example greater than 50%, preferably greater than 60or 75%.

Nucleic acid hybridisation will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridising nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30 degrees C., typically in excess of 37 degrees C., and preferably inexcess of 45 degrees C. Stringent salt conditions will ordinarily beless than 1000 mM, typically less than 500 mM, and preferably less than200 mM. However, the combination of parameters is much more importantthan the measure of any single parameter. An example of stringenthybridisation conditions is 65° C. and 0.1×SSC (1×SSC=0.15 M NaCl, 0.015M sodium citrate pH 7.0).

The “polynucleotide” of this invention include RNA, cDNA, genomic DNA,synthetic forms, and mixed polymers, both sense and antisense strands,and may be chemically or biochemically modified or may containnon-natural or derivatised nucleotide bases, as will be readilyappreciated by those skilled in the art. Such modifications include, forexample, labels, methylation, substitution of one or more of thenaturally occurring nucleotides with an analogue, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

The present invention provides recombinant nucleic acids comprising allor part of anyone of SEQ ID NOS:1 to 77. The recombinant construct maybe capable of replicating autonomously in a host cell. Alternatively,the recombinant construct may become integrated into the chromosomal DNAof the host cell. Such a recombinant polynucleotide comprises apolynucleotide of genomic, cDNA, semi-synthetic, or synthetic originwhich, by virtue of its origin or manipulation, 1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature; 2) is linked to a polynucleotide other than that to which it islinked in nature; or 3) does not occur in nature.

Therefore, recombinant nucleic acids comprising sequences otherwise notnaturally occurring are provided by this invention. Although thewild-type sequence may be employed, it will often be altered, e.g., bydeletion, substitution or insertion.

A “Recombinant nucleic acid” is a nucleic acid that is not naturallyoccurring, or which is made by the artificial combination of twootherwise separated segments of sequence. This artificial combination isoften accomplished by either chemical syntheses means, or by theartificial manipulation of isolated segments of nucleic acids, bygenetic engineering techniques. Such is usually done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a desired combination of functions.

cDNA or genomic libraries generated from lymphocytes caused to beinfected with IBDV may be screened as natural sources of the nucleicacids of the present invention, or such nucleic acids may be provided byamplification of sequences resident in genomic DNA or other naturalsources, e.g., by PCR. The choice of cDNA libraries normally correspondsto a tissue source that is abundant in mRNA for the desired proteins.Phage libraries are normally preferred, but other types of libraries maybe used. Clones of a library are spread onto plates, transferred to asubstrate for screening, denatured and probed for the presence ofdesired sequences.

Techniques for nucleic acid manipulation are described generally, forexample, in Sambrook et al., (1989) “Molecular Cloning: a laboratory,manual”. Sambrook, J., Fritsch, E. F. and Maniatis, T. (eds) (1989).Coldspring Harbour Laboratory Press, Coldspring Harbour, N.Y. or Ausubelet al., (1992) “Current Protocols in Molecular Biology”. Ausubel, F. M.,Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. G.and Struhl, K. (1987). John Wiley and Sons, NY. Reagents useful inapplying such techniques, such as restriction enzymes and the like, arewidely known in the art and commercially available from such vendors asNew England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S.Biochemicals, New England Nuclear, and a number of other sources. Therecombinant nucleic acid sequences used to produce fusion proteins ofthe present invention may be derived from natural or syntheticsequences. Many natural gene sequences are obtainable from various cDNAor from genomic libraries using appropriate probes. See, GenBank,National Institutes of Health.

Portions of the polynucleotide sequence having at least about eightnucleotides, usually at least about 15 nucleotides, and fewer than about6 kb, usually fewer than about 1.0 kb, from a polynucleotide sequenceencoding IBDV related antibody recombinant fragments are preferred asprobes. The probes may also be used to determine whether mRNA encodingantibody recombinant fragments is present in a cell or tissue andwhether the genomic organisation of the constituent parts of theantibody recombinant fragments are deleted or otherwise damaged.

In further aspects, the invention also includes cloning and expressionvectors containing these DNA sequences, host cells transformed withthese DNA sequences and processes for producing the heavy or lightchains and antibody fragments comprising expressing these DNA sequencesin a transformed host cell.

Identification of Recombinant IBDV Antibodies

According to a further aspect the invention provides a process foridentifying recombinant antibody fragments, which process comprises thesteps:

-   (i) Amplifying nucleotide sequences comprising V_(H) and V_(L)    chains from lymphocytes from a host which has been caused to produce    antibodies against at least IBDV or an IBDV polypeptide or a    fragment thereof;-   (ii) Generating a library comprising amplified nucleotide sequences    from step (i), which library is capable of being screened to    identify V_(H) and or V_(L) regions reactive with at least IBDV or    an IBDV polypeptide or a fragment thereof; and-   (iii) Screening said library and selecting at least a recombinant    antibody fragment that has an affinity for IBDV or an IBDV    polypeptide or a fragment thereof.

According to this method nucleotide sequences encoding V_(H) and V_(L)region fragments are derived from B-lymphocytes from a host, which hasbeen caused to produce antibodies against at least IBDV or an IBDVpolypeptide or a fragment thereof. Selecting such nucleotide sequencesand using them to generate recombinant antibody fragments generates alevel of certainty that the V_(H) and V_(L) chains will be specific forthe IBDV agent that provoked the antibody production in theB-lymphocyte. Moreover, by selecting B-lymphocytes infected with aparticular virus as the source for nucleotide sequences for generatingrecombinant antibody fragments, problems attendant with differentialantigen presentation and hence differential immune responses betweendifferent animal species may be ameliorated.

Any system capable of generating a library comprising amplifiednucleotide sequences from step (i), which library is capable of beingscreened to identify V_(H) and V_(L) chains reactive with at least IBDVor an IBDV polypeptide or a fragment thereof may be used in thedescribed method. Preferably, phage display technology is used togenerate and screen the library such technology.

Phage displayed recombinant antibody libraries offer a number ofadvantages over other systems in that they allow expression ofconformational epitopes and enable the use of strong selectionprocedures for the isolation of strain specific antibodies. Theisolation of IBDV specific recombinant antibody fragments from chickenshas the advantage of obtaining antibodies from the normal host. Inaddition, they offer technical advantages over antibodies from othermammalian hosts like mouse as the V region genes in chickens are flankedby constant regions and can easily be amplified by PCR using a singleprimer set. Large libraries of diverse antibody combining sites can beexpressed in E. coli by bacteriophage and can be easily screened.

Phage display is a technique for the expression or ‘display’ of apeptide or protein on the surface of a filamentous phage. This isaccomplished by the insertion of a gene or gene fragment in a phagesurface protein gene. Provided that the reading frame is correct andthat the insert does not interfere with the essential functions of thesurface protein, the insert will result in a fusion protein on the phagesurface. If the peptide is well exposed on the phage surface it will beavailable to act as a ligand, enzyme, immunogen or otherwise activelyparticipate in a biochemical process. The insertion of randomoligonucleotide sequences such as those derived from B-lymphocytes froma host, which has been caused to produce antibodies against at leastIBDV or an IBDV polypeptide or a fragment thereof, provides a means ofconstructing extensive peptide libraries that may be screened to selectpeptides with specific affinities or activities against IBDV molecules.

Separation of phage particles expressing different peptide inserts inthe phage surface protein may be accomplished by any method known in theart. One such method of affinity selection is a method called panning.This strategy allows one to test a myriad of structures for optimalfunction without detailed information about the relationship between thefunction and the structure.

Filamentous phage are ideal as in vitro selection vehicles because theyhave small genomes in which large libraries, consisting of a number ofdifferent genes or gene fragments, are easily constructed. The viralparticles (virions) are stable to potential elution conditions such aslow pH and they accumulate to high titers (10¹² ml⁻¹) so that everyclone in a gene library can be well represented. Some commonly usedphage particles are M13, fd, and f1. The phage have ten different genesof which the two that have been used in phage display are gene III(gill) and gene VIII (gVIII). Gene III encodes a protein at the proximalend of the phage, protein III (pIII). Protein III is required forinfection of E. coli and binds to the end of the pili of the bacteria.Gene VIII encodes a major coat protein, protein VIII (pVIII), and istherefore present in approximately 2700 copies in comparison with geneIII that is present in 3-5 copies depending on the phage used.

Phage display technology can be performed in two different ways,polyvalent phage display and monovalent phage display. In both methodsthe insertion is usually done near the amino-terminal end between theamino- and carboxy-terminal domains. In polyvalent phage display, smallforeign DNA fragments are inserted into the phage surface protein gene.In this method the peptide is expressed in multiple copies on the phageproduced, i.e. the peptide is expressed in all copies of the geneproduct. In polyvalent display the fusion using pIII cannot have a toolarge insert since it will affect the infectivity of the phage. Fusionusing pVIII is even more limited by the size of the insert, since largerpeptides than 6-10 amino acids will probably interfere with thepackaging process. For monovalent phage display, the gene fusion isconstructed in a phagemid where DNA fragments of varying sizes areinserted in the phage surface protein gene. A phagemid is a plasmid withthe intergenic sequence from the phage that is used and the phage originof replication, but it lacks all other phage genes and can therefore notgive rise to new phage. The intergenic sequence contains the packingsequence used for phage construction. The virions are produced afterinfection of the cells harbouring the phagemid with a helper phage. Thehelper phage provide for those functions and genes the phagemid lacksand also contains the wild type gene for the surface protein. The helperphage is packaging-deficient, meaning that the intergenic region of thegenome is not as efficient as in the phagemid. The phagemid is thentransferred into a bacterial host (eg E. coli). Selection by antibioticresistance will result in only bacteria that have acquired the phagemidgrowing. The number of clones that are required to cover the wholegenome is dependent of the size of the gene insert. While the helperphage supplies wild-type protein in large excess, only approximately 10%of the phagemid population will display one copy of the fusion protein.This system removes the problem of a protein fusion affecting the phagepacking properties and functions in the case where all of the proteinshave an insertion. Insertion of the amber stop codon TAG is commonlyinterposed between the foreign gene and the gene used for the fusion.The stop codon is suppressed in suitable strains to allow phageproduction and allows for easy production of soluble protein by transferto a non-suppressor strain.

According to a highly preferred form of the method described herein thelibrary is generated using a phagemid system. The efficacy of such asystem is largely dependant on the phagemid vector selected and used.Numerous phagemid vectors are commercially available: Amersham PharmaciaBiotech, Sweden; Maxim Biotech, USA; Mediators Diagnostika, Austria;Mobitec, Germany; New England Biolabs, USA; Stratagene, USA. Desirably,however, the selected vector enables the direct sequential ligation ofheavy and or light chain nucleotide sequences. By using such a vector, ahigher diversity of recombinant antibodies may be generated.

A particularly preferred phagemid vector that may be used in thedescribed method is pCANTAB-link. This vector is based on pCANTAB 5E.However, it encodes a polypeptide linker region (Gly4Ser)3 flanked bytwo multiple cloning regions. PCR fragments for V_(H) or V_(L) can besequentially ligated into unique restriction sites upstream (SfiI, NcoI,AscI, PstI and XbaI) or downstream (SalI, EcoRV and NotI) of the linker.This vector eliminates the inefficient and problematic PCR assemblystep, replacing it with sequential ligation of V_(H) and V_(L) chainsdirectly into the vector. Using this vector much higher diversities ofrecombinant antibody scFv libraries can be generated by sequentialligation of V_(H) and V_(L) PCR fragments into pCANTAB-link vector thanby using pCANTAB 5E. The presence of unique restriction sites betweenthe polypeptide linker and the variable fragments also offers the optionfor easy downstream modifications of libraries/clones such as convenientshuffling of V_(H) and V_(L) fragments or the exchange of differentpolypeptide linkers (for generation of diabodies etc.). The vector canbe used for the construction of scFv libraries of any animal species.

Advantages of the highly efficient pCANTAB-link vector for libraries ofhigh diversity compared with pCANTAB 5E may be summarised as follows:

-   -   (i) Compatible with PCANTAB 5E system.    -   (ii) Option of two different cloning strategies:        -   (a) either direct sequential ligation of V_(H) and V_(L) PCR            fragments        -   (b) or ligation of PCR assembly products (used like pCANTAB            5E).    -   (iii) Contains additional unique restriction enzyme sites for        subcloning and screening.    -   (iv) Contains region for polypeptide linker (G₄S)₃ (vector        without linker is also available).    -   (v) Facilitates easy replacement of different polypeptide        linkers or shuffling of V_(H) and V_(L) chains to generate new        combinatorial libraries.    -   (vi) Can be used to construct scFv libraries of any species.

Therefore in a preferred form of the above method the recombinant IBDVantibody fragment is identified according to the following method:

-   -   (i) Lymphocytes are obtained from the spleen of chickens        immunised against an infectious bursal disease strain;    -   (ii) mRNA is extracted from those Lymphocytes and transcribed        into cDNA;    -   (iii) V_(H) and V_(L) chain genes are then amplified and        purified;    -   (iv) Purified V_(H) and V_(L) chains are then joined together        via the use of a synthetic linker to yield scFv fragments;    -   (v) The scFv fragments are then cloned into an expression vector        and transformed into a suitable host;    -   (vi) With the aid of a helper phage, recombinant phage specific        for IBDV are selected by panning against a plate coated with        IBDV antigens; and    -   (vii) Phage specific for IBDV antigens are eluted off and grown        up individually.

In an even more preferred form of the invention, steps (iv) and (v) arecombined and a pCANTAB-link vector is used as the expression vector.Thus Purified V_(H) and V_(L) chains are ligated directly into apCANTAB-link vector which contains a synthetic linker to yield scFvfragments. The vector is then transformed into a suitable host.

After, the nucleotide sequences of V_(H) and V_(L) chains are clonedinto the vector a library of recombinant antibody fragments isgenerated. Techniques for inserting such vectors into cells areconventional, for example, transformation, electroporation, protoplastfusion and transfection are examples of well-known methods. The hostcells that may be used for this step in the method may be bacterial (forexample E. coli), fungi, algae, mammalian cells or any other prokaryoteor eukaryotic cell. Desirably the host cell is E. coli.

The recombinant antibody fragments of interest can be selected bytechniques known to persons skilled in the art. Such techniques includethose based on affinity interaction. Standard procedures use eitherantigens coated directly or indirectly (e.g using streptavidin) ontoplastic surfaces (plates or immunotubes) or antigens that arebiotinylated and coupled to strepavidin-coated paramagnetic beads.Selections can be carried out with whole cells or even living organisms.Usually, the antigens are incubated with phage display libraries andspecifically bound phage are eluted after each round. Desirably, theantibody of interest is detected by the technique of panning, which isknown to persons skilled in the art.

Preparation of Recombinant or Chemically Synthesized IBDV RelatedRecombinant Antibody Nucleic Acids; Vectors, Transformation, Host Cells

Once the amino acid constitution for a recombinant antibody fragment isknown, that fragment may be reproduced by any means known in therecombinant DNA art. It should be appreciated that one of thesignificant advantages of using recombinant antibody fragments overtraditional antibody preparation techniques is that such antibodies canbe produced in large volume using standard protein productiontechniques.

Any IBDV related recombinant antibody nucleic acid specimen, in purifiedor non-purified form, can be utilised as the starting nucleic acid oracids for the preparation of recombinant antibody fragment(s).

Functional gene fragments utilised herein may be extracted as mRNA fromany tissue sample, such as blood, tissue material (eg B lymphocytes) andthe like and converted to cDNA by reverse transcription by a variety oftechniques such as that described by Maniatis, et. al. in MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., p 280-281,1982). If the extracted sample has not been purified, it may be treatedbefore amplification with an amount of a reagent effective to open thecells, or animal cell membranes of the sample, and to expose and/orseparate the strand(s) of the nucleic acid(s). This lysing and nucleicacid denaturing step to expose and separate the strands will allowamplification to occur much more readily.

PCR is one such process that may be used to amplify IBDV relatedrecombinant antibody gene sequences. This technique may amplify, forexample, DNA or RNA, including messenger RNA, wherein DNA or RNA may besingle stranded or double stranded. In the event that RNA is to be usedas a template, enzymes, and/or conditions optimal for reversetranscribing the template to DNA would be utilised. In addition, aDNA-RNA hybrid that contains one strand of each may be utilised. Amixture of nucleic acids may also be employed, or the nucleic acidsproduced in a previous amplification reaction described herein, usingthe same or different primers may be so utilised.

The specific nucleic acid sequence to be amplified, i.e., thepolymorphic gene sequence, may be a fraction of a larger molecule or canbe present initially as a discrete molecule, so that the specificsequence constitutes the entire nucleic acid. It is not necessary thatthe sequence to be amplified is present initially in a pure form; it maybe a minor fraction of a complex mixture, such as contained in wholehuman DNA.

A double-stranded fragment may be obtained from the single-strandedproduct of chemical synthesis either by synthesizing the complementarystrand and annealing the strands together under appropriate conditionsor by adding the complementary strand using DNA polymerase with anappropriate primer sequence.

Large amounts of the polynucleotides of the present invention may alsobe produced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eucaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian or plant or othereucaryotic cell lines.

Polynucleotides of the invention may be incorporated into a recombinantreplicable vector for introduction into a prokaryotic or eucaryotichost. Such vectors may typically comprise a replication systemrecognized by the host, including the intended polynucleotide fragmentencoding the desired polypeptide, and will preferably also includetranscription and translational initiation regulatory sequences operablylinked to the polypeptide encoding segment. Expression vectors mayinclude, for example, an origin of replication or autonomouslyreplicating sequence (ARS) and expression control sequences, a promoter,an enhancer and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, and mRNA stabilizing sequences.Secretion signals may also be included where appropriate, which allowthe protein to cross and/or lodge in cell membranes, and thus attain itsfunctional topology, or be secreted from the cell. For example, arecombinant antibody fragment may be expressed with a bacterial leadersequence at the N-terminus capable of driving export of the protein tothe periplasmic space. There, the various domains of the recombinantantibody molecules may fold into functionally active proteins.

Such vectors may be prepared by means of standard recombinant techniqueswell known in the art and discussed, for example, in Sambrook et al.,1989 supra or Ausubel et al. 1992 supra.

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host. Examples of workablecombinations of cell lines and expression vectors are described inSambrook et al., 1989 or Ausubel et al., 1992. Many useful vectors areknown in the art and may be obtained from such vendors as Stratagene,New England Biolabs, Promega Biotech, and others. Promoters such as thetrp, lac and phage promoters, tRNA promoters and glycolytic enzymepromoters may be used in prokaryotic hosts. Useful yeast promotersinclude promoter regions for metallothionein, 3-phosphoglycerate kinaseor other glycolytic enzymes such as enolase orglyceraldehyde-3-phosphate dehydrogenase, enzymes responsible formaltose and galactose utilization, and others. Vectors and promoterssuitable for use in yeast expression are further described in Hitzemanet al., EP 73,675A. Appropriate non-native mammalian promoters mightinclude the early and late promoters from SV40 or promoters derived frommurine Moloney leukaemia virus, mouse tumour virus, avian sarcomaviruses, adenovirus II, bovine papilloma virus or polyoma. In addition,the construct may be joined to an amplifable gene (e.g., DHFR) so thatmultiple copies of the gene may be made. For appropriate enhancer andother expression control sequences.

While such expression vectors may replicate autonomously, they may alsoreplicate by being inserted into the genome of the host cell, by methodswell known in the art.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells that express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc.; b) complement auxotrophic deficiencies, or supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribedin vitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection, or the vectors can be introduceddirectly into host cells by methods well known in the art, which varydepending on the type of cellular host, including electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; infection (where the vector is an infectiousagent, such as a retroviral genome); and other methods. The introductionof the polynucleotides into the host cell by any method known in theart, including, inter alia, those described above, will be referred toherein as “transformation.” The cells into which have been introducednucleic acids described above are meant to also include the progeny ofsuch cells.

Thus the present invention provides host cells transformed ortransfected with a nucleic acid molecule of the invention. Preferredhost cells include bacteria, yeast, mammalian cells, plant cells, insectcells, and human cells in tissue culture. Illustratively, such hostcells are selected from the group consisting of E. coli Pseudomonas,Bacillus, Streptomyces, yeast, CHO, R1.1, B-W, L-M, COS 1. COS 7, BSC1,BSC40, BMT10, and Sf9 cells.

Also provided are mammalian cells containing an IBDV antibodypolypeptide encoding DNA sequence and modified in vitro to permit higherexpression of IBDV related antibody polypeptides by means of ahomologous recombinational event.

The general methods for construction of the vector of the invention,transfection of cells to produce the host cell of the invention, cultureof cells to produce the antibody of the invention are all conventionalmolecular biology methods. Likewise, once produced, the recombinantantibody fragments of the invention may be purified by standardprocedures of the art, including cross-flow filtration, ammoniumsulphate precipitation, affinity column chromatography, gelelectrophoresis and the like.

Thus, the present invention also provides methods for preparing an IBDVrelated antibody polypeptide comprising: (a) culturing a cell asdescribed above under conditions that provide for expression of the IBDVrelated antibody polypeptide; and (b) recovering the expressed IBDVrelated antibody polypeptide. This procedure can also be accompanied bythe steps of: (c) chromatographing the polypeptide using any suitablemeans known in the art; and (d) purifying the polypeptide by for examplegel filtration.

The present invention also provides for host cells transformed with twoor more expression vectors of the invention, the first vector containingan operon encoding a V_(H) chain derived polypeptide and the secondcontaining an operon encoding a V_(L) chain derived polypeptide. The twovectors may contain different selectable markers but, with the exceptionof the V_(H) and V_(L) chain coding sequences, are preferably identical.This procedure provides for equal expression of V_(H) and V_(L) chainpolypeptides. Alternatively, a single vector may be used which encodesboth V_(H) and V_(L) chain polypeptides. The coding sequences for theV_(H) and V_(L) chains may comprise cDNA or genomic DNA or both. In apreferred embodiment of this aspect of the invention at both vectorsprovide leader sequences capable of directing the expressed proteins outof the cell, most preferably into the periplasm where disulphide bondformations may occur.

Many uses for antibodies, which have been produced using the disclosedmethods, are contemplated, including diagnostic and therapeutic uses.

Diagnostic Use and Detection of IBDV

The present invention also provides the above antibody fragments,detectably labeled, as described below, for use in diagnostic methodsfor in vitro or in vivo detection of IBDV.

-   -   (i) IBDV Related Recombinant Antibody Polypeptide Fragments

The recombinant antibody fragments of the present invention may beemployed in any known antibody associated assay method. For example, therecombinant antibody fragments of the present invention are useful forimmunoassays that detect or quantitate IBDV in a sample. For examplethey may be employed in competitive binding assays, direct and indirectsandwich assays, or immunoprecipitation assays and immunohistochemistryassays.

An immunoassay for IBDV will typically comprise incubating a biologicalsample in the presence of a detectably labeled recombinant antibodyfragment capable of binding to IBDV and detecting the labeled antibodywhich is bound in a sample. Various clinical immunoassay procedures aredescribed in Immunoassays for the 80s, A. Voller eds, University Park,1981.

Thus in an embodiment of the diagnostic uses of IBDV related recombinantantibody fragments, the antibody fragment or a biological sample may beadded to nitrocellulose, or other solid support that is capable ofimmobilizing cells, cell particles or soluble proteins. The support maythen be washed with suitable buffers followed by treatment with thedetectably labeled recombinant antibody fragment. The solid phasesupport may then be washed with the buffer a second time to removeunbound antibody fragments. The amount of bound label on said solidsupport may then be detected by conventional means.

By “solid phase support” or “carrier” is intended any support capable ofbinding antigen or antibodies. Well-known supports or carriers, includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toIBDV or. Thus, the support configuration may be spherical, as in a bead,or cylindrical, as in the inside surface of a test tube, or the externalsurface of a rod.

Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports include polystyrene beads. Those skilled in the artwill know many other suitable carriers for binding recombinant antibodyfragments or antigen, or will be able to ascertain the same by use ofroutine experimentation.

For diagnostic applications, the recombinant antibody fragment typicallywill be labelled directly or indirectly with a detectable moiety. Thedetectable moiety can be any one, which is capable of producing, eitherdirectly or indirectly, a detectable signal. Any method known in the artfor separately conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain et al.,J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem.30:407 (1982).

The recombinant antibody fragment can be fused to a short immunogenicpeptide sequence which is detected by an antibody directed against it.

Enzymes which can be used to detectably label the IBDV-specificantibodies of the present invention include, but are not limited to,malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase.

By radioactively labeling the recombinant antibody fragments, it ispossible to detect IBDV through the use of a radioimmunoassay (RIA)(see, for example, Work, T. S., et al., Laboratory Techniques andBiochemisty in Molecular Biology, North Holland Publishing Company, N.Y.(1978). The radioactive isotope can be detected by such means as the useof a gamma counter or a scintillation counter or by autoradiography.Isotopes which are particularly useful for the purpose of the presentinvention are: ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I or ¹³¹I and preferably, ¹²⁵1.

It is also possible to label the recombinant antibody fragments with afluorescent compound. When the fluorescent labeled recombinant antibodyfragment is exposed to light of the proper wave length, its presence canthen be detected due to fluorescence. Among the most commonly usedfluorescent labelling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, 2-phthaldehydeand fluorescamine.

The recombinant antibody fragments can also be detectably labeled usingfluorescence-emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the MF-specific antibody usingsuch metal chelating groups as diethylenetriaminepentaacetic acid orethylenediamine-tetraacetic acid.

The recombinant antibodies also can be detectably labeled by coupling toa chemiluminescent compound. The presence of the chemiluminescentlylabeled antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areisoluminol, theromatic acridinium ester, imidazole, acridinii salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the recombinantantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

Detection of the recombinant antibody may be accomplished by ascintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorometric methods which employ asubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

For the purposes of the present invention, IBDV which is detected by theabove assays may be present in a biological sample. Any samplecontaining IBDV can be used. Preferably, the sample is a tissue extractor homogenate, allantoic fluid, or any biological fluid such as, forexample, blood, serum, lymph, urine, inflammatory exudate, cerebrospinalfluid, and the like and fixed tissue (like in formalin fixed andparaffin embedded blocks) and tissue impression smears. However, theinvention is not limited to assays using only these samples, it beingpossible for one of ordinary skill in the art to determine suitableconditions which allow the use of other samples.

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and providing the combination of labeledantibodies of the present invention to such a specimen. The antibody (orfragment) is preferably provided by applying or by overlaying thelabeled antibody (or fragment) to a biological sample.

Therefore according to an embodiment of the invention there is provideda method for detecting presence of IBDV in a sample comprisingcontacting said sample with a recombinant antibody fragment whichspecifically binds to an IBDV antigen, comprising a V_(H) region and aV_(L) region, said V_(H) region having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:76 to 112, and said V_(L) regionhaving an amino acid sequence selected from the group consisting of SEQID NOS:113 to 154, and determining binding of said recombinant antibodyfragments to IBDV antigen in said sample as a determination of IBDVpresence in said sample.

It will be appreciated that by varying the V_(H) and V_(L) regionsemployed in the antibody fragments it is possible to alter the affinityand hence possible use to which said antibodies may be put. For example,SEQ ID NOS:157 to SEQ ID NOS:195 show varying degrees of specificity forIBDV strains. Thus different fragments may be employed in differingdiagnostic applications. In particular, SEQ ID NOS:159, 160, 187, 195,158, 167, 156, 162, 171, 172, 173, 184 and 174 (CRAb's 15, 5, 12, 20,24, 66, 83, 96, 149, 151, 154, 174 and 176) are reactive with all mostall or all IBDV strains tested in the Examples. Such fragments may beemployed to identify the presence of IBDV in a sample. Phage antibodylike CRAb 20 (SEQ ID NOS:195), that react strongly with denatured IBDVsamples may be used as diagnostic reagents on fixed and/or denatureddiagnostic samples such as in immunohistochemistry of fixed infectedtissue and in protein blotting applications. Further, SEQ ID NOS:192 and163 (CRAb's 11 & 34) are only reactive with Australian IBDV strainstested in the Examples. Such fragments might be used to differentiateAustralian IBDV strains from IBDV strains from closely relatedcountries. Further, SEQ ID NO:176 (CRAb 88) is only reactive with veryvirulent IBDV strains such as CS88 tested in the Example and suchfragments might be used to differentiate very virulent IBDV strains fromall other IBDV strains.

The recombinant antibodies, for example such as described in the exampleherein SEQ ID NO:155 to SEQ ID NO:196 can be packaged into diagnostickits. Diagnostic kits include the recombinant antibodies which may belabelled; alternatively, the recombinant antibodies may be unlabeled andthe ingredients for labelling may be included in the kit. The kit mayalso contain other suitably packaged reagents and materials needed forthe particular antigen or antibody detection, for example, standards, aswell as instructions for conducting the test. Recombinant antibodyfragments are also useful for the affinity purification of IBDV fromrecombinant cell culture or natural sources.

-   -   (ii) IBDV Related Recombinant Antibody Polynucleotide Fragments

Polynucleotides encoding recombinant antibody fragments may also be usedto provide diagnostic analysis. For example, allele specificoligonucleotide primers derived from IBDV related recombinant antibodygene sequences, particular those gene sequences encoding IBDVneutralising recombinant antibodies described herein may be useful indetermining whether an animal is at risk of suffering from an IBDVailment. Alternatively by detecting changes in the transcription of andor translation of polynucleotide sequences described herein it will bepossible to identify whether a particular host is suffering from aparticular IBDV ailment. Therefore through the use of such a procedure,it is possible to determine not only the presence of IBDV but also thedistribution of IBDV in the examined tissue.

According to one detection system recombinant antibody polynucleotidesmay be identified using PCR related technologies. Many different PCRrelated technologies suitable for such use are known in the field. Suchmethodologies are broadly described in Ausubel, F., Brent, R., Kingston,R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. Currentprotocols in molecular biology. Greene Publishing Associates/WileyIntersciences, New York and are incorporated herein by reference.

Primers used in any diagnostic assays derived from the present inventionshould be of sufficient length and appropriate sequence to provideinitiation of polymerisation. Environmental conditions conducive tosynthesis include the presence of nucleoside triphosphates and an agentfor polymerisation, such as DNA polymerase, and a suitable temperatureand pH.

Primers are preferably single stranded for maximum efficiency inamplification, but may be double stranded. If double stranded, primersmay be first treated to separate the strands before being used toprepare extension products. Primers should be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent for polymerisation. The exact length of a primer will depend onmany factors, including temperature, buffer, and nucleotide composition.Oligonucleotide primers will typically contain 12-20 or morenucleotides, although they may contain fewer nucleotides.

The deoxyribonucleotide triphosphates dATP, dCTP, dGTP and dTTP areadded to the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool,which is preferable for, the primer hybridisation.

To the cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein “agent for polymerisation”),and the reaction is allowed to occur under conditions known in the art.The agent for polymerisation may also be added together with the otherreagents if it is heat stable. This synthesis (or amplification)reaction may occur at room temperature up to a temperature above whichthe agent for polymerisation no longer functions. Thus, for example, ifDNA polymerase is used as the agent, the temperature is generally nogreater than about 40° C. Most conveniently the reaction occurs at roomtemperature.

Some other useful diagnostic techniques for detecting the presence ofparticular fragments and or mutations to the fragment genes that encoderecombinant antibody fragments of particular interest include, but arenot limited to: 1) allele-specific PCR; 2) single stranded conformationanalysis (SSCA); 3) denaturing gradient gel electrophoresis (DGGE); 4)RNase protection assays; 5) the use of proteins which recognizenucleotide mismatches, such as the E. coli mutS protein; 6)allele-specific oligonucleotides (ASOs); and 7) fluorescent in situhybridisation (FISH). Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE), heteroduplex analysis (HA) andchemical mismatch cleavage (CMC).

In addition to the above methods recombinant antibody fragment genes andmutants thereof may be detected using conventional probe technology.Using the disclosed portions of the isolated IBDV related recombinantantibody polynucleotide fragments as a basis oligomers of approximately8 nucleotides or more can be prepared, either by excision orsynthetically, which hybridise with the IBDV related recombinantantibody polynucleotides.

The probes for IBDV related recombinant antibody polynucleotides(natural or derived) are a preferably of a length which allows thedetection of these unique sequences. While 6-8 nucleotides may be aworkable length, sequences of 10-12 nucleotides are preferred, and about20 nucleotides would be optimal. Preferably, these sequences will derivefrom regions which lack heterogeneity. These probes can be preparedusing routine methods, including automated oligonucleotide syntheticmethods.

For use as probes, complete complementarity is desirable, though it maybe unnecessary as the length of the fragment is increased. For use ofsuch probes as diagnostics, the biological sample to be analysed istreated, if desired, to extract the nucleic acids contained therein. Theresulting nucleic acid from the sample may be subjected to gelelectrophoresis or other size separation techniques; alternatively, thenucleic acid sample may be dot blotted without size separation.

When probes are used to detect the presence of the target sequences, thebiological sample to be analysed, such as tissue homogenate, may betreated, if desired, to extract the nucleic acids. The sample nucleicacid may be prepared in various ways to facilitate detection of thetarget sequence; e.g. denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the samplenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniquesknown in the art.

Sample nucleic acid and probe are incubated under conditions thatpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the sample. The region of the probesthat is used to bind to the sample can be made completely complementaryto the targeted region. Therefore, high stringency conditions aredesirable in order to prevent false positives. However, conditions ofhigh stringency may be used only if the probes are complementary toregions of the chromosome that are unique in the genome. The stringencyof hybridisation is determined by a number of factors duringhybridisation and during the washing procedure, including temperature,ionic strength, base composition, probe length, and concentration offormamide. Under certain circumstances, the formation of higher orderhybrids, such as triplexes, quadraplexes, etc., may be desired toprovide the means of detecting target sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labelled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand that islabelled, either directly or indirectly. Suitable labels, and methodsfor labelling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labelledmoiety.

Two detection methodologies that are particularly effective, work on theprinciple that a small ligand (such as digoxigenin, biotin, or the like)is attached to a nucleic acid probe capable of specifically binding IBDVrelated antibody polynucleotides. The small ligand is then detected. Inone example, the small ligand attached to the nucleic acid probe mightbe specifically recognized by an antibody-enzyme conjugate. For example,digoxigenin may be attached to the nucleic acid probe. Hybridisation isthen detected by an antibody-alkaline phosphatase conjugate that turnsover a chemiluminescent substrate. In a second example, the small ligandmay be recognized by a second ligand-enzyme conjugate that is capable ofspecifically complexing to the first ligand. A well-known example is thebiotin-avidin type of interactions.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting IBDV related antibodypolynucleotides. Thus, in one example to detect the presence of IBDVrelated antibody polynucleotides in a cell sample, more than one probecomplementary to IBDV related antibody polynucleotides is employed andin particular the number of different probes is alternatively 2, 3, or 5different nucleic acid probe sequences. In another example, to detectthe presence of mutations in the IBDV related antibody polynucleotidesgene sequence in an animal, more than one probe complementary to IBDVrelated antibody polynucleotides is employed where the cocktail includesprobes capable of binding to an allele-specific mutation identified inpopulations of animals with alterations in IBDV related antibodypolynucleotides. In this embodiment, any number of probes can be used,and will preferably include probes corresponding to the major genemutations identified as predisposing an animal to the IBDV infection.

In a highly preferred embodiment, screening techniques based onhybridisation to probes, particularly a plurality of probes thatcorrespond to allele-specific mutations use probes immobilized to solidsubstrates as described above, for example in the form of DNA arrays onsilicon substrates (DNA chips).

The probes or primers described herein can be packaged into diagnostickits. Diagnostic kits include the probe DNA, which may be labelled;alternatively, the probe DNA may be unlabeled and the ingredients forlabelling may be included in the kit. The kit may also contain othersuitably packaged reagents and materials needed for the particularhybridisation protocol, for example, standards, as well as instructionsfor conducting the test.

Therapeutic Use

The present invention also provides therapeutic, pharmaceutical orprophylactic compositions, which may take any suitable form, foradministration to an animal to treat that animal against IBDV relatedailments. It also provides methods for the administration of theantibodies fragments, either labelled or unlabelled, to an animal.

According to the invention the therapeutic, pharmaceutical orprophylactic composition will preferably comprise at least a recombinantantibody fragment as described herein and a pharmaceutically acceptablecarrier. While any of the described fragments that have a bindingaffinity for IBDV or a IBDV polypeptide or fragment thereof may be usedin the compositions, preferably the fragment will possess a propertywhich facilitates inhibition or neutralization of IBDV. Most preferablythe fragment will possess the capacity to neutralise virus infection. Asan example of a highly preferred form of the invention the fragment isidentified by SEQ ID NOS:157, 160, 191, 194, 192, 159, 195, 189, 188,158, 161, 163, 190, 156, 162, 172 and 184 (CRAbs 3, 5, 8, 9, 11, 15, 20,22, 23, 24, 33, 34, 52, 83, 96, 154 and 174) each of which display aninhibition or neutralisation ability for IBDV infection.

Where the recombinant antibody fragment(s) is to be administered to a ananimal it is preferably in a form suitable for administration e.g. byinjection or infusion, for example by bolus injection or continuousinfusion. Where the product is for injection or infusion, it may takethe form of a suspension, solution or emulsion in an oily or aqueousvehicle and it may contain adjuvants and or formulatory agents such asacceptable carriers, excipients or stabilizers.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the immunogen and also as a lymphoid systemactivator that nonspecifically enhances the immune response [Hood etal., in Immunology, p. 384, Second Ed., Benjamin/Cummings, Menlo Park,Calif. (1984)].

Acceptable carriers, excipients or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA, sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

Therapeutic formulations of the recombinant antibody fragments may beprepared for by mixing the recombinant antibody fragments having thedesired degree of purity with optional physiologically acceptablecarriers, excipients, or stabilizers (Remington's PharmaceuticalSciences, 16th edition, Osol, A., Ed., [1980]), in the form oflyophilised cake or aqueous solutions.

The recombinant antibody fragments may also be administered either asindividual therapeutic agents or in combination with other therapeuticagents. For example the recombinant antibody fragments of this inventionmy be utilized in combination with other monoclonal antibodies or otherantibody fragments and regions or with lymphokines or hemopoietic growthfactors, etc., which serve to increase the number or activity ofeffector cells which interact with the antibodies.

An example of therapeutic application of recombinant antibodies is whenthe recombinant antibody is complexed with either live vaccine, orvirus. Such CRAb.IBDV complex for example can be given to chickens (orany other host) as an alternative vaccination approach by variety ofmeans. Recombinant antibodies can also be complexed with an antigen sucha peptide or protein or a whole inactivated virus, or other biologicallyactive molecules. Such CRAb.antigen complex for example can beadministered to animals to induce higher immune responses; or fordelivery to a particular site to achieve a variable biological effect.

The fragments may also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-[methylmethacylate] microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

The fragments to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution. Thefragments ordinarily will be stored in lyophilized form or in solution.

Therapeutic fragment compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierce-able by a hypodermic injection needle.

The route of fragment administration will accord with known methods,e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems as noted below.The fragments may also be administered continuously by infusion or bybolus injection.

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981) andLanger, Chem. Tech. 12:98-105 (1982) or poly(vinylalcohol)],polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers22:547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON DEPOT.TM. (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for antibody stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release fragment compositions also include liposomallyentrapped fragments. Liposomes containing the antibody are prepared bymethods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad.Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Nat. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal antibody therapy.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the animal. Accordingly, it will benecessary for the therapist to titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect. Atypical daily dosage might range from about 1 mg/kg to up to 10 mg/kg ormore, depending on the factors mentioned above. Typically, the clinicianwill administer fragments until a dosage is reached that achieves thedesired effect. The progress of this therapy is easily monitored byconventional assays.

The present invention further provides the use of a polypeptide orpolynucleotide of the invention, which polypeptide or polynucleotide is,or encode, biologically active in gene therapy. Thus the inventionprovides a method of treating a IBDV disease which method comprisesadministering to said cells a functional recombinant antibodypolypeptide or polynucleotide encoding said polypeptide fragment tosuppress IBDV proliferation in a host.

Thus a recombinant antibody fragment polynucleotide sequence may beintroduced into the cell or host (or live animal) in a vector or asnaked DNA such that the polynucleotide sequence remainsextrachromosomal. In such a situation, the polynucleotide sequence willbe expressed by the cell from the extrachromosomal location. If apolynucleotide sequence is introduced and expressed in a cell carrying amutant IBDV related antibody encoding polynucleotide sequence, thepolynucleotide sequence should encode an IBDV related antibody proteinthat is capable of disturbing IBDV proliferation. More preferred is thesituation where the wild-type polynucleotide sequence is introduced intothe mutant cell in such a way that it recombines with the endogenousmutant polynucleotide sequence present in the cell. Such recombinationrequires a double recombination event that results in the correction ofthe polynucleotide sequence mutation.

Vectors for introduction of genes both for recombination and forextrachromosomal maintenance are known in the art, and any suitablevector may be used. However, replication-incompetent retroviral vectorshave proved safe and effective in recent trials and most of the approvedhuman gene therapy trials to date rely on retroviral vectors. Thus it ispreferred to use retroviral vectors, such as lentiviral vectors,comprising a polynucleotide of the invention and capable of expressing apolypeptide of the invention. Other viral vector systems includeadenoviral vectors and herpes virus vectors.

Methods for introducing DNA into cells such as electroporation, calciumphosphate co-precipitation and viral transduction are known in the art,and the choice of method is within the competence of the person skilledin the art. A further gene transfer technique that has been approved bythe FDA is the transfer of plasmid DNA in liposomes. Suitable liposomecompositions include Lipofectin™.

Gene therapy would be carried out according to generally acceptedmethods. Cells from an animal would be first analysed by the diagnosticmethods described above, to ascertain the production of IBDV relatedrecombinant antibody fragment in a host. A virus or plasmid vector (seefurther details below), containing a copy of an IBDV related recombinantantibody fragment polynucleotide sequence linked to expression controlelements and capable of replicating inside preferably a lymphocyte, isprepared. Suitable vectors are known, such as disclosed in U.S. Pat. No.5,252,479 and PCT published application WO 93/07282. The vector is theninjected into the patient, either locally or systemically. If thetransfected gene is not permanently incorporated into the genome of eachof the targeted cells, the treatment may have to be repeatedperiodically.

Gene transfer systems known in the art may be useful in the practice ofthe gene therapy methods of the present invention. These include viraland nonviral transfer methods. A number of viruses have been used asgene transfer vectors, including papovaviruses, e.g., SV40, adenovirus,vaccinia virus, adeno-associated virus, herpesviruses including HSV andEBV, and retroviruses of avian, murine, and human origin. Most genetherapy protocols have been based on disabled murine retroviruses.

Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate co-precipitation; mechanicaltechniques, for example microinjection; membrane fusion-mediatedtransfer via liposomes; and direct DNA uptake and receptor-mediated DNAtransfer. Viral-mediated gene transfer can be combined with direct invivo gene transfer using liposome delivery, allowing one to specificallydirect the viral vectors to the cells of interest Alternatively, theretroviral vector producer cell line can be injected into thelymphocytes. Injection of producer cells would then provide a continuoussource of vector particles.

In an approach which combines biological and physical gene transfermethods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalisation, and degradation of theendosome before the coupled DNA is damaged.

Liposome/DNA complexes have been shown to be capable of mediating directin vivo gene transfer. While in standard liposome preparations the genetransfer process is non-specific, localised in vivo uptake andexpression have been reported in some tumour deposits, for example,following direct in situ administration.

Best Mode(s) for Carrying Out the Invention

Further features of the present invention are more fully described inthe following non-limiting Figures, Tables and Example. It is to beunderstood, however, that this description is included solely for thepurposes of exemplifying the present invention. It should not beunderstood in any way as a restriction on the broad description of theinvention as set out above.

EXAMPLE

Methods of molecular biology that are not explicitly described in thefollowing examples are reported in the literature and are known by thoseskilled in the art.

General texts that described conventional molecular biology,microbiology, and recombinant DNA techniques within the skill of theart, included, for example: Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Glover ed., DNA Cloning: A PracticalApproach, Volumes I and II, MRL Press, Ltd., Oxford, U.K. (1985); andAusubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G.,Smith, J. A., Struhl, K. Current protocols in molecular biology. GreenePublishing Associates/Wiley Intersciences, New York. McCafferty, J.,Hoogenboom, H. R. and Chiswell, D. J. (eds.), Antibody engineering, apractical approach (1996), Oxford University Press.

Virus Strains

Australian IBDV strain 002/73 described by Firth (1974), AustralianVeterinary Journal 50; 128-130, and vvIBDV strain CS88 [Brown, M.D., etal (1994), J. Gen Virol. 75, 675-680)] were used for hyper-immunizationof chickens and for ELISA. Additional Australian IBDV strains used forELISA included vaccine strain V877, nine classical field strains (06195,K3, M4, R1, T4, A-1, Y-5, N1/99, N2/99) and variant field strains (H-1,01/94, 02/95, 03/95, 04/95 and 08/95) [Sapats and Ignjatovic (2000)Archives of Virology 145; 773-785; Ignjatovic and Sapats; submitted forpublication]. Overseas strains used included classical strains 52/70,1/68, DV86 AND APHIS; variant strains E, A, GLS AND Ga, and veryvirulent strains CS88, DV8, UK661, VB849, 99006, 89163, 91168, 94432,Tsik94, Indo1, Indo6 and Indo10 [Bayliss, C. D. et al (1990) J. Gen.Virol. 71, 1303-1312; Heine, H. G., et al. (1991). J. Gen. Virol 72,1835-1843; Eterradossi et al, (1997) Arch Virol 142, 255-270; Parede etal, submitted for publication].

Immunisation of Chickens

Four three-week-old specific pathogen free (SPF) chickens were immunizedwith Australian vaccine strain 002/73 or vvIBDV strain CS88, aspreviously described [Sapats and Ignjatovic, (2000) Archives of Virology145; 773-785]. Chickens were boosted two weeks later by intramuscularinjection with 0.5 ml of inactivated preparation of 002/73 or CS88(diluted 1/10 using PBS and emulsified with an equal volume of FreundsIncomplete Adjuvant). Four weeks post secondary immunization, chickenswere euthanased and spleens removed for purification of lymphocytes.Titres of immune sera were determined by ELISA.

Preparation of mRNA and cDNA

Spleens from immunized chickens (ELISA antibody titres≧200,000) wereminced through a stainless steel mesh screen with cold PBS and layeredonto an equal volume of Histopaque (Sigma). White blood cells wereseparated by centrifugation for 20 minutes at 700×g and mRNA extractedfrom 2.4×10⁹ cells using QuickPrep mRNA Purification kit (Pharmacia).

Approximately 8 μg of mRNA was used to produce randomly primed cDNA(total volume 100 μl) using a cDNA synthesis kit (Boehringer Mannheim).

PCR Amplification of Heavy and Light Chain Variable Domain Genes

PCR reactions were set up in a total volume of 100 μl containing 5 μl ofcDNA, 10 μl of 10×Pfu buffer (Stratagene), 1 μl dNTPs (25 mM each), 2 μl(6 ng) of each oligonucleotide: HF & HR for the heavy chain variableregion (VH) or LF & LR for the light chain variable region (VL) (Table1). After chilling the tubes on ice, 2 μl of cloned PFU was added(Stratagene) and reactions were cycled 30 times for 45 sec at 94° C., 45sec at 50° C. and 2 min at 72° C. In the final cycle, the extension timeat 72° C. was increased to 10 min. Amplifications were repeated fivetimes each and the final VH and VL products were pooled respectively. VLand VH products of approximately 350 bp and 390 bp were gel purifiedusing a gel extraction kit (Qiagen).

TABLE 1 Sequences of oligonucleotides used for construction of librariesand improved phagmid vectors. Restriction enzyme sites used for cloningare shown underlined. Primer Nucleotide Sequence (5′ to 3′) HFGCCGTGACGTTGGAC HR GAACCGCCTCCACCGGAGGAGACGATGACTTCGG* LFCGGTGGCGGATCGGCGCTGACTCAGCC LR ACCTAGGACGGTCAGGG* Link1GGTGGAGGCGGTTCAGGCGGAGGTGGCTCT Link2 CGATCCGCCACCGCCAGAGCCACCTCCGCCTGA*HF-Sfi ATGTCTATGGCCCAGCCGGCCGTGACGTTGGACG LR-NotAGTTACTGGAGCGGCCGCACCTAGGACGGTCAGGG* LF-SalGGCGGTGGCGGGTCGACAGCGCTGACTCAGCCGTCC TCG HR-XbaGAACCGCCTCCACCATCTAGAGAGGAGACGATGACTTCGG* HF-AscTTAGCTGGGCGCGCCGTGACGTTGGACGAGTC B94CGGCCATGGGGCGCGCCGTCTAGAGCTAAGATATCGC B95GGCCGCGATATCTTAGCTCTAGACGGCGCGCCCCATGGCC GGCT* B99CGCGCCACTGCAGCTCTAGATCCCGGGTCGACAGATATCAGTGC B100GGCCGCACTGATATCTGTCGACCCGGGATCTAGAGCTGCA GTGG* B107TAACTAATTCTAGATGGTGGAGGCGGTTCAGGCGGAGGTG GCTCT B108TATAGATTATGTCGACCCGCCACCGCCAGAGCCACCTCCGCCT* Seq 1GGTTCAGGCGGAGGTGGCTCTGG Seq 2 AGAGCCACCTCCGCCTGAACC*PCR Assembly of Single Chain Fv (scFv) Genes into PCANTAB 5E Vector

Purified VH and VL chains were connected via a synthetic linker(Gly₄Ser)₃ using overlap extension PCR. Purified VH and VL (100 ng each)were used in PCR as described above containing 2 μl (20 ng) of eachlinker primer, Link1 & Link2 (Table 1). After 7 rounds of amplification(94° C. for 1 min, 55° C. for 1 min and 72° C. for 2 min), 2 μl (200 ng)of each primer HF-Sfi and LR-Not were added (Table 1) and an additional24 cycles was performed. Approximately 500 ng of the 750 bp scFv overlapextension product was gel purified and digested sequentially with SfiIand NotI according to the manufacturers instructions (NEB). TheSfiI/NotI digested scFvs were further purified using a PCR purificationkit (Qiagen).

Approximately 300 ng of the digested scFv fragments were ligated with500 ng of SfiI/NotI digested pCANTAB 5E vector (Pharmacia) in a totalvolume 100 μl using the Rapid DNA Ligation Kit (Boehringer Mannheim).Contaminating salts were removed from the ligated DNA using the RapidPCR purification kit (Boehringer Mannheim). DNA was electroporated intoelectrocompetent E. coli TG1 cells with a transformation effeciency ofapproximately 1×10⁹/μg pUC19 DNA. An aliquot was taken to titrate thelibrary size on SOB plates containing 100 μg/ml ampicillin and 2%glucose (SOB-AG) (Sambrook et al., Molecular Cloning: A LaboratotyManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)). The remaining culture was plated out onto SOBAGplates and incubated overnight at 30° C. The resulting lawn of bacterialcells were scraped into 20 ml LB and a 1 ml aliquot diluted with 9 ml2xYT containing 50 μg/ml ampicillin and 2% (w/v) glucose (2xYT-AG)(Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)) and infected with 6×10¹⁰ pfu of M13KO7. After 2 hrs of shakingat 37° C. the cells were collected by centrifugation and resuspended in10 ml 2xYT containing 100 μg/ml ampicillin and 50 μg/ml kanamycin. Theculture was incubated overnight at 37° C. with shaking at 250 rpm. Cellswere pelleted at 1400 g for 15 min and the supernatant, containingphage, was filtered through a 0.45 μm filter. Phage particles wereconcentrated from the supernatant by PEG precipitation (Griffiths etal., 1993, EMBO J. 12, 775-734). The concentration of infectious phageparticles was determined by infecting log phase E. coli strain TG1 withserially diluted phage, incubating at 37° C. for 30 min and plating onSOB-AG plates.

Although high yields of chicken immunoglobulin V_(H) and V_(L) fragmentswere obtained by PCR amplification from spleen derived cDNA, thesubsequent assembly of V_(H) and V_(L) into scFv fragments was highlyinefficient, producing libraries containing only 1.5×10⁵ clones. Theselibraries resulted in the identification of one unique CRAb (CRAb34)using the 002/73 library (FIGS. 6, 7). To improve upon the inefficientassembly and cloning steps, we developed a new vector that enabled thedirect sequential ligation of V_(H) and V_(L) PCR fragments into avector that already encoded a flexible linker region (Gly₄Ser)₃.

Construction of Phagemid Vectors for High Diversity scFv Libraries

The pCANTAB 5E vector was modified in order to acquire additionalrestriction enzyme sites for direct subcloning of individual V_(H) andV_(L) fragments, resulting in 3 different modifications (FIGS. 1, 2).Unique restriction sites (NcoI, AscI, XbaI and EcoRV) were insertedbetween the SfiI and NotI sites of the pCANTAB 5E vector by ligation oftwo partially overlapping oligonucleotides (B94 and B95) (Table 1). Theresulting vector was named pCANTAB-Ascl (FIG. 1). Two partiallyoverlapping oligonucleotides (B99 and B100) (Table 1) were then insertedbetween the AscI and NotI sites of pCANTAB-AscI to introduce additionalunique SmaI and PstI restriction enzyme sites. The resulting vector wasnamed pCANTAB-SmaI (FIG. 1). An oligonucleotide encoding the polypeptidelinker (Gly₄Ser)₃ was constructed by annealing two overlappingoligonucleotides (B107 and B108) (Table 1) and filling in with Klenowpolymerase. The resulting linker fragment was digested with XbaI andSalI to generate the appropriate overhanging ends, and inserted betweenthe XbaI and SalI restriction sites of pCANTAB-SmaI, yielding thephagemid pCANTAB-link (FIG. 1).

All modifications left the SfiI and NotI restriction sites and thereading frame of the parent vector pCANTAB 5E intact. The firstmodification, pCANTAB-AscI, introduced restriction sites AscI, NcoI,XbaI and EcoRV that could be used for subcloning of inserts. The secondmodification produced pCANTAB-Smal, which contains additional uniqueSmaI and PstI restriction sites. The third vector, pCANTAB-link, encodesa flexible linker region (Gly₄Ser)₃, which allows construction offunctional scFv antibodies by sequential insertion of V_(H) and V_(L)fragments on either side of the linker region. This eliminates the needto connect the VH and VL at the PCR level. All three modified vectorscan still be used in the same way as pCANTAB 5E for subcloning ofSfiI-NotI scFv fragments, but in addition they can also be used tosequentially ligate V_(H) and V_(L) fragments compatible with a range ofdifferent restriction sites.

Sequential Ligation of V_(H) and V_(L) Fragments into pCANTAB-linkVector

The vector pCANTAB-link was used for the direct sequential cloning ofV_(H) and V_(L) PCR fragments into multiple cloning sites on either sideof the flexible linker region (Gly₄Ser)₃ to generate functional scFvclones. The restriction fragments AscI-XbaI were chosen for the ligationof V_(H) fragments upstream of the linker region and SalI-NotIdownstream of the linker to generate scFv. This was achieved in thefollowing manner.

V_(H) and V_(L) chains were amplified as described previously usingmodified primers HF-Sfi/HR-Xba and LF-Sal/LR-Not (Table 1). V_(H) andV_(L) fragments were gel purified and digested with AscI/XbaI andSalI/NotI, respectively (NEB). The pCANTAB-link vector was digested witheither AscI/XbaI (for cloning of VH) or SalI/NotI (for cloning of V_(L))and the V_(H) and V_(L) chains cloned respectively, creating 2intermediate plasmids pCANTAB-link-H and pCANTAB-link-L. Afterpropagation of these plasmids in E. coli DH5α cells, V_(L) was clonedinto the SalI/NotI site of pCANTAB-link-H and VH was cloned into theAscI/XbaI site of pCANTAB-link-L. The ligated DNA was electroporatedinto electrocompetent E. coli TG1 cells and recombinant phage producedas described above.

A range of published immunoglobulin variable regions from chicken andmouse were assessed for the absence of six-base cutter restrictionenzyme sites that could be used for cloning. This assessment revealedthe XbaI and SalI sites to be suitable for direct subcloning (of eitherSfiI-XbaI, AscI-XbaI or SalI-NotI fragments). If XbaI or SalI sites werepresent within the variable immunoglobulin fragments, differentrestriction enzymes that yield compatible ends for cloning could beincorporated in the ends of the PCR primers used for amplification ofthe immunoglobulin variable regions. Compatible ends for XbaI (T′CTAG,A)can be generated with NheI (G∝CTAG,C) and SpeI (A′CTAG,T), andcompatible ends for SalI (G′TCGA,C) can be generated with XhoI(C′TCGA,G). A useful feature of the pCANTAB-link is the facility toeasily replace different linkers (for example to generate diabodies ormultimers) and to swap V_(H) and V_(L) domains for the construction ofnew combinatorial libraries from existing clones.

Selection Procedure for Phage Antibodies

Immunotubes (Maxisorb, Nunc) were coated overnight at room temperaturewith rabbit anti-IBDV sera (4 ml, diluted 1/500 in 50 mM sodiumcarbonate buffer, pH 9.6). After washing with PBS containing 0.1% Tween20 (PBS-T), 002/73 virus (4 ml, diluted 1/200 in PBS containing 5% FCS)was captured at 37° C. for 1 hour. Tubes were blocked for 1 hr with PBScontaining 5% skim milk and panning was carded out according toinstructions provided with the pCANTAB 5E expression module (Pharmacia),with some modifications. Briefly, 8 ml of the PEG precipitated phage wasmixed with 7 ml of 5% skim milk, and 3 ml was poured onto the blockedtubes. After a 2 hr incubation at 37° C. the tubes were washed 20 timeswith PBS, followed by 20 washes with PBS-T. Log phase E. coli TG1 cells(4 ml) were added to the tubes and incubated at 37° C. to allowinfection by phage. Aliquots were plated onto SOB-AG to determine thetitre of phage. The remaining culture was infected with helper phageM13K07 as described previously and subjected to an additional 3 roundsof panning. In later experiments the number of washes between rounds 1,2, 3 and 4 of panning was reduced to 5, 10, 20 and 40 washes with PBS-T,respectively.

Construction and Selection of scFv Libraries from Immunised Chickens inpCANTAB 5E and the Modified pCANTAB-Link Vector

The scFv recombinant antibody libraries in the pCANTAB 5E vectorproduced 1.5×10⁵ clones with almost 100% of the clones carrying a scFvinsert. The library was subjected to two different panning procedures.In the first procedure, phage were panned three times and washed 40times after each adsorption. After each panning step, 88 clones werescreened for binding in an ELISA against 002/73. After the first, secondand third pan, 2%, 28% and 81% of clones respectively, were ELISApositive All clones isolated from the third round of panning appeared tobe identical by DNA sequencing and were designated chicken recombinantantibody 34 (CRAb34). One ELISA negative scFv, designated CRAb0, waschosen from the original unpanned library to serve as a negativecontrol. The second panning procedure consisted of 4 panning steps usingless stringent conditions, with the number of washes between panningsbeing reduced to 5, 10, 20 and 40 after the first, second, third andfourth panning steps, respectively. Despite a reduction in the number ofwashing steps it was not possible to isolate additional antigen positiveCRAbs other than those identical to CRAb34. To overcome the limitedlibrary diversity, new libraries were prepared using the modified vectorpCANTAB-link as described above.

The same chicken lymphocyte mRNA used for construction of the pCANTAB 5Elibrary was also used for the construction of the scFv library in thepCANTAB-link vector. The V_(H) and V_(L) genes were cloned sequentiallyinto the pCANTAB-link vector. Two separate intermediate libraries wereconstructed, one containing the H chain the other containing the Lchain. Cloning the H or L chain first made no difference to thediversity of the library. The final library containing both the H and Lchains yielded approximately 7.5×10⁷ clones and upon superinfection withhelper phage gave rise to approximately 4.7×10¹² phage particles. Phagewere subjected to three rounds of panning with 5, 10, 20 and 40 washesafter each round, respectively. The binding capacity of (combined) phagewas examined after each round of panning using ELISA. With eachsuccessive round of panning, an increase in the ELISA absorbance wasobserved indicating an enrichment of IBDV specific clones (results notshown).

Expression of scFvs

(i) Expression of Phage Displayed Antibodies:

Individual colonies were inoculated into 500 μl of 2xYT-AG and grown at30° C. overnight with shaking at 250 rpm. Aliquots of 50 μl weretransferred to 500 μl of 2xYT-AG containing M13KO7 helper phage.Cultures were shaken at 37° C. for 2 hrs at 150 rpm and then centrifugedat 1000 g for 15 mins. The bacterial pellets were resuspended in 2xYTcontaining 100 μg/ml ampicillin and 50 μg/ml kanamycin grown at 30° C.overnight with shaking at 250 rpm. Cells were pelleted and thephage-containing supernatant (˜400 μl) removed for analysis in ELISA.For large scale screening against IBDV strains cultures were scaled upto 10 ml.

(ii) Expression of Soluble Antibodies:

ELISA positive phage were used to infect a non-suppressor strain of E.coli (HB2151) according to the instructions provided by Pharmacia withsome minor modifications. Briefly, 2 μl of phage supernatant was used toinfect log phase E. coli HB2151 cells and the cultures were plated outonto SOB-AG containing 100 μg/ml Naladixic Acid (SOB-AGN). Singlecolonies were inoculated into 2xYT-AG and cultures were grown shakingovernight at 30° C. Aliquots of overnight culture were diluted 1/10 intofresh SB medium containing 100 μg/ml ampicillin and 2% (w/v) glucose(SB-AG) and incubated shaking at 30° C. for 2.5 hrs. Cells were pelletedand resuspended in fresh SB-A medium containing 1 mM IPTG and shaken for6 hours at 30° C. to induce expression of soluble scFv protein (Sab).Cells were pelleted and the periplasm (containing soluble antibodies)extracted using mild osmotic shock (Alvi A Z, et al (1999) Hybridoma 18,413-421). For neutralization studies, large-scale stocks of solubleantibodies were produced and dialysed extensively against PBS.

Characterisation of rec Ab Clones by ELISA

Phage ELISA: For detection of binding to IBDV, phage were subjected toELISA in which IBDV was captured onto microtitre plates in the samemanner as described for panning, but in 100 μl volumes. For the initialscreening, IBDV was diluted 1/100, but a dilution of 1/20 was used forall subsequent experiments. Bound phage were detected using anti-M13 HRPconjugate (Pharmacia) and absorbances at 405 nm were recorded after theaddition of ABTS [2, 2-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid)diammonium salt].

ELISA with Sabs: ELISA plates were coated with rabbit-anti-IBDV IgGovernight at room temperature. IBDV antigen diluted in 2% skim milk wasthen added and incubated for 1 hr at room temperature. After washingSabs, diluted 1/10 in 2% skim milk, were added and incubated for 1 hr atroom temperature. Bound Sabs were detected using anti-E tag monoclonalantibody (Pharmacia), followed by addition of goat anti mouse IgG-HRPconjugate (Biorad). Absorbances were recorded as for the phage ELISA.

ELISA of Phage Against Different IBDV Strains

Approximately 1,000 individual clones obtained from the third and fourthrounds of panning were screened in ELISA for reactivity against 002/73or CS88 virus. Of the 1,000 clones examined, 46% reacted positively inan ELISA. Around 290 ELISA positive clones were randomly chosen andexamined for their reactivity against a panel of Australian and overseasIBDV strains, first as phage displayed antibodies and subsequently assoluble antibodies. In the initial screen, the viral antigen used forcoating of ELISA plates was diluted 1/100. When antigen was diluted 1/20in subsequent ELISAs, all phage showed higher levels ofcross-reactivity, making it more difficult to differentiate clones onthe basis of ELISA reactivity (Table 2). The majority of CRAbs werecross-reactive, both as phage and soluble antibodies and included CRAbs24, 96, 83, 15, 5, 22, 12, 151, 18, 176, 174, 154, 66, 149, and 20.Other CRAbs such as CRAb11 and 34 appeared to be specific for Australianstrains, while CRAb 88 appeared specific for vvIBDV. A number ofinteresting differences were observed when comparing the same CRAbexpressed as either on the surface of phage or as soluble antibody, inparticular CRAbs 33, 8 and 9. Depending on the expression system, theseCRABs could differentiate between Australian and overseas strains, withCRAb33 and CRAb9 being specific for Australian strains when expressed asa soluble antibody, while the reverse situation was true for CRAb8.

DNA Sequence Determination and Analysis

Phagemid DNA was isolated using the Qiagen midi prep (low copy plasmidprocedure). Each scFv construct was sequenced using the S4, S6sequencing primers (Pharmacia), which were complementary to the vectorsequence. Two additional primers Seq 1 and Seq 2 (Table 1) were usedwhich were complementary to the linker sequence. Nucleotide sequenceswere determined using the Big Dye Terminator Ready Reaction Kit inconjunction with the 377 XL automated DNA sequencer (AppliedBiosystems). Resulting sequences were aligned using the CLUSTAL-Xmultiple sequence alignment program version 1.81 (Thompson, J. D., et al(1997) Nucleic Acids Research 25, 4876-4882). DNA sequence analyses wereperformed using analysis programs on the Internet accessed via theAustralian National Genomic Information Service (ANGIS) or PC basedprograms (DNASIS, PROSIS).

TABLE 2 ELISA reactivity pattern of different CRAbs (as phage displayed= p or as soluble antibodies = s) with a panel of IBDV strains. [++++,+++; ++, + = optical density greater than 1.1, 0.7-1.1, 0.3-0.7, and0.1-0.3, respectively; − = optical density less than 0.1; NT = nottested]. Tested under saturating conditions; antigen (bursa homogenate1/20), Sab 1/10, phage diluted 1/5 using 10% skim milk (final 2%).002/73 V877 06/95 K3 M4 R1 T4 N1/99 N2/99 A-1 Y5 H-1 01/94 02/95 03/9504/95 08/95 52/70 1/68 Var E Cs88 Neg 002/73 CRAb 34p +++ +++ ++ + −− + + + + + − − − − − − − − − − − derived CRAb34s ++++ ++++ +++ +++ ++++ +++ +++ + ++ +++ ++ + ++ ++ − − − + − − − CRAb24p ++++ ++++ +++ ++++++ ++ +++ +++ +++ +++ +++ +++ +++ ++ ++ − − +++ ++ − ++ − CRAb24s ++++++++ ++++ ++++ +++ +++ ++++ ++++ +++ ++++ +++ +++ +++ +++ +++ − − ++++++ − ++ − CRAb3p ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++++++ ++++ ++++ ++++ − − ++++ ++++ − ++++ − CRAb3s ++++ ++++ +++ +++ ++++ +++ +++ ++ +++ +++ ++ ++ ++ ++ + + + + − ++ − CRAb33p ++++ ++++ +++++++ ++++ ++++ +++ ++++ ++++ +++ ++++ +++ +++ +++ +++ − − ++++ +++ − ++++− CRAb33s ++++ ++++ +++ +++ ++ ++ +++ ++++ +++ +++ +++ +++ ++ ++ +++ − −− − − − − CRAb96p +++ +++ +++ ++++ +++ +++ +++ ++++ ++++ ++++ ++++ ++++++ +++ +++ − − +++ +++ − +++ − CRAb96s ++++ ++++ ++++ ++++ +++ ++ ++++++++ +++ +++ ++++ +++ ++ ++ +++ − − ++ +++ − ++ − CRAb83p ++++ ++++ ++++++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ − − ++++++++ − ++++ − CRAb83s ++++ ++++ ++++ ++++ +++ +++ ++++ ++++ +++ +++ +++++++ +++ +++ +++ − − ++ +++ − ++ − CRAb15p ++++ ++++ ++++ ++++ ++++ ++++++++ ++++ +++ ++++ ++++ ++++ ++++ ++++ ++++ − − ++++ ++++ − ++++ −CRAb15s ++++ +++ ++++ ++++ +++ ++ +++ ++++ +++ +++ ++++ +++ +++ ++ +++ −− ++ +++ − ++ − CRAb5p ++++ +++ +++ ++++ +++ ++ +++ +++ +++ +++ +++ ++++ ++ ++ − − ++ +++ − ++ − CRAb5s ++++ ++++ ++++ ++++ +++ +++ ++++ ++++++++ ++++ ++++ ++++ ++++ +++ +++ + − +++ ++++ − ++++ − CRAb11p ++++ ++++++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++ ++ ++ ++ ++ − − − − −CRAb11s ++++ ++++ ++++ ++++ +++ +++ ++++ ++++ ++++ ++++ +++ ++++ ++ +++++++ ++ ++ − − − − − CRAb21p +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++++ ++ ++ ++ ++ ++ ++ ++ − ++ − CRAb21s +++ ++++ ++++ +++ ++ + +++ ++ ++++++ ++ − − − − − − + + − + − CRAb23p ++++ +++ ++++ ++++ +++ ++++ ++++++++ ++++ ++++ ++++ ++++ ++++ +++ ++++ − − +++ +++ − +++ − CRAb23s ++++++++ +++ ++ + + +++ ++ ++ ++ ++ + + + + − − + + − ++ − CRAb22p ++++ +++++++ +++ +++ +++ +++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ − − +++ ++++ −+++ − CRAb22s ++++ +++ ++++ +++ ++ ++ +++ +++ +++ ++ +++ +++ ++ +++ +++− − ++ ++ − ++ − CRAb52p ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++++++ ++++ ++++ ++++ ++++ ++++ − − ++ ++ − ++ − CRAb52s ++++ +++ +++++ + + ++ ++ ++ ++ ++ +++ ++ ++ ++ − − − − − + − CRAb8p ++++ ++++ ++++++ ++ ++ +++ ++ +++ +++ +++ ++++ +++ +++ +++ − − − − − − − CRAb8s ++++++++ ++++ ++++ +++ +++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ +++ − − +++++ − +++ − CRAb7p ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++++++ − − − − − ++ ++ − ++ − CRAb7s ++++ ++++ ++++ +++ +++ +++ +++ ++++++++ ++++ ++ − − + − − − − − + − CRAb12p ++++ ++++ ++++ ++++ ++++ ++++++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ − ++++− CRAb12s ++++ ++++ ++++ ++++ ++ ++ +++ ++++ +++ +++ +++ +++ +++ +++ +++++ ++ ++ ++ − ++ − CRAb20p +++ +++ ++++ +++ +++ ++ +++ +++ ++ ++ +++ ++++ +++ ++ + ++ ++++ +++ ++ ++++ − CRAb20s ++++ +++ ++++ ++++ +++ +++++++ + + + + + + +++ +++ + − +++ ++++ − ++++ − CRAb9p +++ +++ ++++ ++++++ +++ +++ + +++ ++ + ++ + ++ ++ − ++ ++ ++ − ++ − CRAb9s ++++ ++++++++ ++++ +++ ++ ++++ +++ +++ ++++ +++ +++ +++ +++ +++ + − − − − − −CRAb0p − − − − − − − − − − − − − − − − − − − − − − CRAb0s − − − − − − −− − − − − − − − − − − − − − − Mab 9-6 ++++ ++++ ++++ ++++ ++++ ++++ ++++++++ ++++ ++++ ++++ ++++ ++ ++++ ++++ + + ++++ ++++ − ++++ − CS88 CRAb151p ++++ ++++ ++++ ++++ ++++ ++++ ++++ NT NT NT NT NT NT +++ +++ NT +++++ +++ + +++ − derived CRAb 151s ++++ ++++ ++++ ++++ ++++ ++++ ++++ NTNT NT NT NT NT ++++ ++++ NT ++++ ++++ ++++ + ++++ − CRAb 28p − − − − − −− NT NT NT NT NT NT − − NT − ++++ +++ − ++++ − CRAb 28s − − − − − − + NTNT NT NT NT NT + − NT − ++++ ++++ − ++++ − CRAb 39p − − − − − − − NT NTNT NT NT NT − − NT − ++ ++ − ++++ + CRAb 39s + − + − − − − NT NT NT NTNT NT + + NT + + + − ++ − CRAb 35p − − − − + − − NT NT NT NT NT NT ++ ++NT ++ − − − ++ − CRAb 35s + + ++ − ++ ++ − NT NT NT NT NT NT ++++ ++++NT ++++ + + − ++++ − CRAb 18p ++++ ++++ ++++ ++++ − − ++++ NT NT NT NTNT NT − + NT − ++++ ++++ − ++++ − CRAb 18s ++++ ++++ ++++ ++++ − − ++++NT NT NT NT NT NT − + NT − ++++ ++++ − ++++ − CRAb 45p ++++ ++++ ++++ −++++ ++ − NT NT NT NT NT NT ++++ +++ NT +++ ++++ ++ ++ ++++ + CRAb 45s++ +++ ++ + + − + NT NT NT NT NT NT ++ + NT + ++ + − +++ − CRAb 27p + ++++ + ++ ++ + NT NT NT NT NT NT +++ +++ NT +++ +++ +++ − ++++ − CRAb27s + + +++ + ++ ++ + NT NT NT NT NT NT ++++ ++++ NT ++++ ++ ++ − ++++ −CRAb 50p − − − − − − − NT NT NT NT NT NT − − NT − + + − ++++ − CRAb 50s− − − − − − − NT NT NT NT NT NT − − NT − +++ +++ − ++++ − CRAb 176p ++++++++ ++++ ++++ ++++ ++++ ++++ NT NT NT NT NT NT ++++ ++++ NT +++ ++++++++ + ++++ − CRAb 176s ++++ ++++ ++++ ++++ ++++ ++++ ++++ NT NT NT NTNT NT ++++ ++++ NT ++++ ++++ ++++ + ++++ − CRAb 30p − + − − − − − NT NTNT NT NT NT − − NT − ++ + − ++++ − CRAb 30s + − − − − − − NT NT NT NT NTNT − − NT − + + − +++ − CRAb 88p − − − − − − − NT NT NT NT NT NT − − NT− − − − ++++ − CRAb 88s − − − − − − − NT NT NT NT NT NT − − NT − − − −+++ − CRAb 62p +++ ++++ ++++ − ++ + − NT NT NT NT NT NT ++++ ++ NT +++++ + + ++++ + CRAb 62s ++ ++ ++ − + − − NT NT NT NT NT NT ++ − NT + ++− − ++ − CRAb 38p − + +++ − − − − NT NT NT NT NT NT − + NT − ++++ ++++ −+++ − CRAb 38s − − ++++ − − − − NT NT NT NT NT NT − − NT − ++++ ++++ −++++ − CRAb 174p +++ ++++ ++++ ++++ +++ +++ ++++ NT NT NT NT NT NT +++++ NT ++ +++ +++ − +++ − CRAb 174s ++++ ++++ ++++ ++++ ++++ ++++ ++++NT NT NT NT NT NT ++++ ++++ NT ++++ ++++ ++++ − ++++ − CRAb 154p ++++++++ ++++ ++++ ++++ ++++ ++++ NT NT NT NT NT NT ++++ ++++ NT ++++ ++++++++ ++ ++++ − CRAb 154s ++++ ++++ ++++ ++++ ++++ ++++ ++++ NT NT NT NTNT NT ++++ ++++ NT +++ ++++ ++++ + ++++ − CRAb 66p ++++ ++++ ++++ ++++++++ ++++ ++++ NT NT NT NT NT NT ++++ ++++ NT ++++ ++++ ++++ ++ ++++ −CRAb 66s ++++ ++++ ++++ ++++ +++ ++++ ++++ NT NT NT NT NT NT +++ ++++ NT+++ ++++ ++++ + ++++ − CRAb 32p + ++ +++ ++ − − ++ NT NT NT NT NT NT − +NT − +++ +++ − ++++ − CRAb 32s ++++ ++++ ++++ ++++ − − ++++ NT NT NT NTNT NT + − NT − ++++ ++++ − ++++ − CRAb 149p ++++ ++++ ++++ ++++ ++++++++ ++++ NT NT NT NT NT NT ++++ ++++ NT +++ ++++ ++++ + +++ − CRAb 149s++++ ++++ ++++ ++++ ++++ ++++ ++++ NT NT NT NT NT NT ++++ ++++ NT ++++++++ ++++ + ++++ − CRAb 37p +++ ++++ ++++ ++++ − − ++++ NT NT NT NT NTNT − + NT − ++++ ++++ − ++++ − CRAb 37s ++++ ++++ ++++ ++++ − − ++++ NTNT NT NT NT NT − − NT − ++++ ++++ − ++++ − CRAb 19p ++++ ++++ ++++ +++++++ +++ ++++ NT NT NT NT NT NT +++ +++ NT − +++ +++ − ++ − CRAb 19s ++++++++ ++++ ++++ ++++ ++++ ++++ NT NT NT NT NT NT ++++ ++++ NT − ++++ ++++− ++++ − CRAb 29p − − − − − − − NT NT NT NT NT NT − − NT − ++++ ++++ −++++ − CRAb 29s − − − − − − − NT NT NT NT NT NT − − NT − ++++ ++++ −++++ − CRAb 26p ++++ ++++ ++++ ++++ − − ++++ NT NT NT NT NT NT − − NT −++++ +++ − ++++ − CRAb 26s − − − − − − − NT NT NT NT NT NT − − NT − − −− + −

Nucleotide sequencing of 60 clones revealed 42 unique CRAbs. Analignment of the deduced amino acid sequences of the CRAbs is shown inFIG. 5 along with that of the negative control CRAb0. Threecomplementarity regions and four framework regions were identified ineach of the H and L chains. Eight of the CRAbs (CRAb24, 3, 33, 96, 83,15, 5, and 19) possessed an identical H chain, which was the same asthat found in CRAb34 (except for two additional amino acids L & D whichoccur by virtue of the pCANTAB-link vector) but all varied in their Lchain sequences. Despite having the same H chains, these 9 CRAbs showsignificant differences in reactivity against IBDV strains (Table 2).For example, CRAb24, 96, 83, 15 and 5 appeared to be reactive with mostIBDV strains tested, whilst CRAb11 and 34 were specific for Australianstrains. The nucleotide and amino acid sequence of 35 individual V_(H)chains (1-35) and 42 individual V_(L) chains (36-77) is shown in FIG. 3and FIG. 4.

Binding Activity in ELISA

Antigen binding activity of CRAbs was determined in ELISA usinghomologous antigen e.g. either 002/73 or CS88 (Table 2). As shown inTable 2, some CRAbs reacted with all IBDV strains (CRAb151, 154, 66, 149and 176). CRAbs 28, 29 and 50 recated only with classical IBDV strainsfrom Europe and did not react with any of Australian IBDV strains, orthe USA variants. CRAbs 9, 11, 33, 34 and 52, as soluble antibodies,reacted only with Australian IBDV strains. CRAb88 reacted only with veryvirulent strain CS88 and did not react with any classical or variantstrains (Table 2). Additionally it was shown that CRAb88 reacted inELISA only with all known very virulent IBDV strains, the results ofwhich (with selected strains only) are shown in Table 3. This indicatesthat CRAb88 is specific for vvIBDV strains and can be used fordifferential diagnosis of vvIBDV. Some CRAbs showed differences inantigen specificity when expressesd as phage and soluble antibodies.Such differences are shown in FIG. 6 with CRAb34, as an example; CRAb34as phage antibody binds only to 002/73 and V877 antigens, whereas assoluble antibody CRAb34 binds to all Australian IBDV strains, albeit toa lesser degree (FIG. 6 & FIG. 7). Binding titres of 29 CRAbs for 002/73antigen are shown in Table 4.

TABLE 3 Specificity of CRAb88 for very virulent IBDV strains in ELISAVirus Virus type Mab 9-6 CRAb154 CRAb88 GLS Variant − +++ − Ga + +++ −VarE + ++ − VarA − ++ DV86 Classical ++++ ++++ − 52/70 ++++ ++++ − 1/68++++ ++++ − APHIS ++++ ++++ − Cu1M Classical NT* ++ − TadForte vaccinesNT ++++ − 228E NT ++ − D78 NT +++ − Bursine NT + − PBG98 NT +++ − DV86Very virulent +++ ++++ +++ UK661 +++ ++++ +++ CS88 ++++ ++++ +++ VB849+++ ++++ +++ 99006 NT ++++ +++ 89163 NT ++++ ++++ 91168 NT ++++ ++++94432 NT ++++ +++ Tasik94 ++++ ++++ +++ Indo1 ++++ ++++ +++ Indo6 ++++++++ +++ Indo10 ++++ ++++ +++CRAbs Ability to Compete with Polyclonal Immune Chicken Sera for Bindingto IBDV Antigen in ELISA

Chicken immune IBDV sera was titrated first to determine the dilutionthat would give 80-90% of maximum binding in a capture ELISA. Forcompetition ELISA, soluble CRAbs, in log₂ dilution, were incubated witha constant amount of 002/73 or CS88 antigen. After one hour, 100 μl ofthis reaction mixture was added to ELISA plates coated with rabbitanti-IBDV sera. After incubation for 1 hour, a constant amount ofchicken immune IBDV sera was added, followed by addition ofgoat-anti-chicken IgG-HRP. After incubation of 1 hour at roomtemperature, substrate was added and absorbance measured at 405 nm. Asshown in FIG. 1 , many CRAbs competed effectively against anti-002/73antisera for binding to 002/73 antigen indicating that CRAbs and immuneIBDV chick sera recognise the same epitopes on the IBDV antigen.Negative control CRAb0 did not compete with immune sera (FIG. 8).

CRAbs were able to differentiate type of IBDV strains that chickens havebeen exposed to, using their immune sera (FIGS. 9 & 10). As shown inFIG. 9, only CRAbs specific for Australian IBDV strains (CRAb11, 34, 33and 52) competed with anti-002/73 antisera for binding to 002/73antigen, whereas CRAbs specific for overseas strains (CRAb28, 29 and 88)did not inhibit binding. Similarly in FIG. 10 is shown that CRAbsspecific for Australian strains (CRAb11, 34, 33 and 52) did not competewith anti-CS88 antisera for binding to CS88 antigen, however, CRAbs 28,29 and 88 did compete. Altogether these results indicate that CRAb88 canbe used to differentiate expose of chickens to very virulent IBDVstrains, after active infection has waned and when only immune sera isavailable. Furthermore that exposure to different types of IBDV, such asvaccinal and field exposure, can be differentiated in immune sera usingselected CRAbs.

Virus Neutralizing Activity In Vitro

The neutralizing activity of all CRAbs was assessed in vitro using thefollowing method. Antibody, in 1092 dilutions, was incubated withapproximately 30-50 median tissue culture infective doses (CID₅₀) ofGT101 IBDV strain [Fahey et al (1991) Avian Diseases 35, 365-373] for 1hr at 37° C. The antibody/virus mixture was then added to the suspensionof freshly prepared secondary chicken embryo fibroblasts and incubatedat 37° C. in the presence of 3% CO₂. Four to 5 days later cellmonolayers in wells of tissue culture plates were examined forcytopathic effect visually. Complete absence of a cytopathic effect inall replicates (quadruplicate for each CRAb dilution) was taken toindicate virus neutralization. The results in Table 4 show that 19 CRAbswere able to neutralize IBDV in vitro. Ten CRAbs had titres higher than128 compared with a negative control scFv (CRAb0) which was not able toneutralize GT101 virus. Eight other antibodies showed lower neutralizingtitres of between 8-64.

TABLE 4 ELISA and virus neutralisation (VN) titres of different CRAbsexpressed as soluble antibodies, obtained in vitro, in ovo and in vivoELISA VN titre % VN in IBDV binding in CEFs ovo of 100 VN in vivo withCRAb protein titre with with EID₅₀ of 002/73 BursavacL Origin CRAbspecificity*  002/73^(#) GT101^(#) BursavacL 100 CID₅₀ 10 CID₅₀ 100CID₅₀ 002/73 0 None 0 0 0 none none library 3 VP3 10,000 0 71 5 VP210,000 6,400 100 partial none 7 VP2 20,000 0 75 none 8 VP2 40,000 320075 partial partial 9 VP2 30,000 >10,240 71 none none 11 VP240,000 >10,400 50 none 12 VP2 16,000 >128 0 none none 15 VP2 80,0001,024 80 partial partial 20 VP3 10,000 12,800 67 none none 21 VP2 2,5608 0 22 VP2 40,000 160 100 none 23 VP2 80,000 160 80 none none 24 VP22,500 64 89 none complete partial 33 VP2 6,400 16 86 partial completenone 34 VP2 2,500 16 80 partial complete partial 52 VP2 10,000 32 100partial partial 83 VP2 5,600 64 86 partial complete partial 96 VP210,000 64 100 partial complete none CS88 18 VP2 >5,120 8 0 partiallibrary 62 VP3 2,560 0 0 66 VP2 >5,120 0 0 88 VP2 512 0 0 149 VP2 >5,1200 0 151 VP2 >5,120 0 0 none 154 VP2 5,120 0 83 none complete none 174VP2 >5120 >128 0 partial 176 VP2 >5,120 0 0 Mab 9-6 VP2 160 5,120 100complete complete Chicken anti- VP2, VP3 100,000 64,000 100 completecomplete BVVirus Neutralizing Activity In Ovo

All CRAbs from Table 4 were incubated with 100 median egg infectivedoses (EID₅₀) of IBDV vaccine strain BursavacL and injected into theallantoic cavity of 10-day-old embryonating chicken eggs. Embryos wereincubated for 7-8 days at 37° C. Embryos were inspected daily for deathsdue to IBDV. At the end of incubation period the number of survivingembryos were counted and expressed as % of neutralisation in comparisonto virus control where all embryos were dead. Results in Table 4 showthat the majority of CRAbs neutralised IBDV in ovo.

Virus Neutralizing Activity In Vivo

CRAbs as well as polyclonal chicken anti-IBDV sera (anti-BV) andmonoclonal antibody 9-6 specific for VP2 of IBDV (Mab 9-6) (Fahey K J,et al (1991) Avian Diseases 35, 365-373) were incubated with 10, 100 or1000 median chick infective doses (CID₅₀) of BursavacL, or 100 CID₅₀ of002/73 strain and injected subcutaneously into the nape of neck of dayold or two-week-old specific pathogen-free (SPF) chickens to assessCRAbs ability to neutralise virus infectivity in vivo. At 7 and 14 daysafter injection bursa were collected from 6 chicks and bursa to bodyweight determined. If bursae were reduced in size in comparison to thenon-infected controls (regressed) at both day 7 and day 14 afterinoculation it was taken to mean that virus was not neutralised. Ifbursae were normal in size (not regressed) at both day 7 and day 14after inoculation it was taken to mean that virus was completelyneutralised. If bursae were normal size (not regressed) at day 7 andregressed at day 14 after inoculation it was taken to mean thatneutralisation of virus was partial. The results obtained with CRAbs areshown in Table 4 and with selected CRAbs in FIG. 11. As shown in Table4, some CRAbs (CRAb8, 15, 34, 52 and 83) were able to neutralise 100CID₅₀ of both BursavacL and 002/73, whereas CRAb5, 33 and 96 were ableto neutralise only 002/73 and CRAb24 to neutralise only BursavacL andnot 002/73. In FIG. 11 is shown the time course of partialneutralisation of 100 CID₅₀ of BursavacL by CRAbs 8, 34 and 52; allthree CRAbs neutralised BursavacL at day 7 post infection whereas viruswas released from the complex and induced bursal regression at day 14post infection as did the polygonal immune anti-IBDV sera (FIG. 11).Ability of CRAbs to neutralize IBDV was dependent on the amount ofinfectious virus used. As shown in Table 4, many CRAbs were able toneutralise completely a low dose of BursavacL, being 10 CID₅₀, howeverwith higher dose of virus (100 CID₅₀) neutralisation was either partialor no neutralisation was achieved. In FIG. 12 is shown the ability ofthe constant amount of CRAb8 to neutralise various doses of BursavacL.Also in FIG. 12 is shown that in 2-week-old chickens higher doses ofBursavacL can be neutralised with the same amount of CRAbs unlike inday-old chicks. Overall these results showed that CRAbs are able to holtthe onset of IBDV infection in chickens by complexing the IBDV virusparticle in a manner identical to that observed for polyclonal chickensera (Whitfill et al., (1995) Avian Diseases 39, 687-699; Haddad et al.,(1997), Avian Diseases 41, 882-889)

Virus Neutralizing Activity Following Inoculation of IBDV. CRAb Mixtureinto 18-Day-Old Embryonating Chicken Eggs

Eighteen-day-old embryonated SPF chicken eggs were inoculated witheither 100 EID₅₀ or BursvacL or 100 EID₅₀ of BursvacL mixed with CRAb8or phosphate buffered saline. Chickens were hatched from these eggs,placed into positive pressure isolation units for containment ofinfection, and bursa collected at various times after infection. Bursato body ratio was determined and bursal tissue frozen, sectioned andused in an immunofluorescence assay with Mab 9-6 to determine thepresence of virus in tissue of these chickens. Only results of bursalregression observed in these chickens is shown in FIG. 13. Chicks thatreceived only BursavacL all had regressed bursa at day 6 after hatch,whereas in those inoculated with Bursavacl.CRAb8 mixture first signs ofregression was visible at day 13 after hatch. These results showed thatCRAb8 was able to holt the onset of infection when given as a mixturewith IBDV to 18-day-old embryonating eggs in a manner similar to thatobserved for polyclonal chicken sera (Jeurissen et al. (1998),Immunology 95, 494-500.

Mapping of Antibody Binding

Viral proteins of 002/73 or CS88 were solubilized with 2%n-octylglucoside and used in immunoprecipitation experiments todetermine the antigenic target of all CRAbs. Each CRAb was incubatedwith either 002173 or CS88 solubilised antigen and the complex wasimmunoprecipitated using anti E-tag monoclonal antibody followed byProtein A-Sepharose. Immunoprecipitated proteins were detected bySDS-PAGE and Western blotting, using monoclonal antibodies 9-6 (fordetection of VP2) and 17-80 (for detection of VP3) [Fahey et al., (1991)Avian Diseases 35, 365-373]. An example of this specificimmunoprecipitation by CRAbs is shown in FIG. 14, in which CRAb34 wasshown to be specific for VP2. As shown in Table 4 the majority of CRAbswere specific for VP2 protein, whereas only three CRAbs 3, 20 and 62were specific for VP3. The negative control antibody, CRAb0 did not bindeither VP2 or VP3 (Table 4 and FIG. 14).

It is to be understood that the above examples are included solely forthe purposes of exemplifying the present invention. They should not beunderstood in any way as a restriction on the broad description of theinvention as set out above.

1. A recombinant antibody fragment having a binding specificity for anIBDV antigenic determinant, the recombinant antibody fragment comprisinga variable region comprising: a) the heavy chain variable region of SEQID NO:92; b) the light chain variable region of SEQ ID NO:125; or c) CDR1, 2, and 3 of a) and b); and wherein the recombinant antibody fragmentbinds vvIBDV strain CS88.
 2. A recombinant antibody fragment comprisinga V_(H) region of SEQ ID NO:92 and a V_(L) region of SEQ ID NO:125. 3.The recombinant antibody fragment according to claim 1 conjugated with,or attached to other antibodies or parts thereof.
 4. The recombinantantibody fragment according to claim 1 which are multivalentmonospecific and comprise at least two single chain antibody fragmentsbound to each other by a connecting structure which protein is not anatural immunoglobulin and wherein at least one of said recombinantantibody fragments has a specificity for an IBDV antigenic determinant.5. The recombinant antibody fragment according to claim 1 wherein V_(H)domains and the V_(L) domains are linked.
 6. The recombinant antibodyfragment(s) according to claim 5 wherein the linker is (Gly₄Ser)₃. 7.The recombinant antibody fragment according to claim 1 wherein thefragment is capable of distinguishing at least two IBDV strains.
 8. Therecombinant antibody fragment according to claim 1 having a differentialbinding affinity capable of distinguishing at least two IBDV strains. 9.The recombinant antibody fragment according to claim 1 capable ofbinding one or more of the group of IBDV selected from: Australian IBDVstrain 002/73; vaccine strain V877; classical field strains 06/95, K3,M4, R1, T4, N 1/99, N2/99, A-1 or Y-5; variant field strains 01/94,02/95, 03/95, 04/95 or 08/95; classical overseas strains 52/70 and 1/68;variant strain E and vvIBDV strain CS88.
 10. The recombinant antibodyfragment according to claim 1 derived from a host that has been causedto produce antibodies against IBDV or an IBDV polypeptide or fragmentthereof.
 11. The recombinant antibody fragment according to claim 10wherein the host is of avian origin.
 12. The recombinant antibodyfragment according to claim 10, wherein the host is a fowl.
 13. A kitfor diagnosis of IBDV strain comprising an antibody fragment orfragments according to claim
 1. 14. The recombinant antibody fragmentsaccording to claim 1 initially derived from nucleotide sequencesencoding heavy and light chain variable regions produced inB-lymphocytes from a host that has been caused to produce antibodiesagainst at least IBDV or an IBDV polypeptide or a fragment thereof. 15.A therapeutic composition comprising at least a recombinant antibodyfragment according to claim 1 and a pharmaceutically acceptable carrier.16. A method of treating a IBDV disease comprising administering afunctional recombinant antibody polypeptide of claim 1 to suppress IBDVproliferation in a host.
 17. A recombinant antibody fragment comprisinga heavy chain variable region of SEQ ID NO:92, wherein the antibodyfragment binds vvIBDV strain CS88.
 18. A recombinant antibody fragmentcomprising a light chain variable region of SEQ ID NO:125, wherein theantibody fragment binds vvIBDV strain CS88.
 19. The recombinant antibodyfragment according to claim 1, wherein the recombinant antibody fragmentdifferentially binds one or more very virulent IBDV strains compared toclassical IBDV strains or virulent IBDV strains.
 20. The recombinantantibody fragment according to claim 17, wherein the recombinantantibody fragment differentially binds one or more very virulent IBDVstrains compared to classical IBDV strains or virulent IBDV strains. 21.The recombinant antibody fragment according to claim 18, wherein therecombinant antibody fragment differentially binds one or more veryvirulent IBDV strains compared to classical IBDV strains or virulentIBDV strains.
 22. A recombinant antibody fragment according to claim 10wherein the fowl is a chicken, turkey, guinea fowl, duck, or goose.